NAV1.7 BINDERS

BACKGROUND OF THE INVENTION
Field of the Invention

The present invention relates to antibodies and antigen-binding fragments thereof that bind the human voltage-gated sodium channel Nav1.7a protein subunit (Nav1.7 binders). In particular, the present invention relates to Nav1.7 binders comprising a heavy-chain immunoglobulin single variable domain (ISVD or VHH).

Description of Related Art

Nav1.7α subunit belongs to a family of nine voltage-gated sodium channels that play crucial roles in the electrical conductance of skeletal muscles (Nav1.4α), cardiac muscles (Nav1.5α), central (Nav1.1α, Nav1.2α, Nav1.3α and Nav1.6α) and peripheral (Nav1.1α, Nav1.6α, Nav1.7α, Nav1.8α and Nav1.9α) neurons. Nav1.7α is mainly expressed on different types of afferent fibres of the peripheral nervous system and is essential to the firing of action potentials by boosting subthreshold stimuli (Dib-Hajj & Waxman 2015 Pain 156: 2406). Extensive genetic evidence in mice and men suggests that Nav1.7 is necessary and non-redundant in pain and olfactory pathways (reviewed by Dib-Hajj et al. 2013 Nat Rev Neurosci. 14: 49). Interestingly, a large and diverse body of naturally occurring toxins acts on voltage-gated sodium channels, including Nav1.7α (reviewed by Deuis et al., 2017 Neuropharmaco DOI10.1016/j.neuropharm.2017.04.014). Nav1.7α has been one of the most hotly pursued targets in the field of chronic pain where there is a large unmet need (reviewed by de Lera Ruiz & Kraus 2015 J Med Chem 58: 7093). Marketed painkillers like local anaesthetics effectively target voltage-gated sodium channels but suffer from undesired side effects prohibiting widespread use in chronic pain indications. Recent efforts to generate more selective Nav1.7α small molecule inhibitors or modified peptide toxins have failed to deliver a marketed drug so far. Attempts to generate selective anti-Nav1.7α biologicals were not reproducible (Lee et al 2014 Cell 157:1393; Liu et al. 2016 F1000Res 5:2764; and many patents).

Four consecutive similar domains, DI to DIV (FIG. 1), make up the nearly 2,000 amino acids large Nav1.7α channel. Each domain has six transmembrane helices (51 to S6 in bottom panel FIG. 1 connected by extracellular loops (ECLs) and intracellular loops (ICLs) (respectively solid and dotted lines in bottom panel FIG. 1. Two small (S1-52 and S3-S4) and one larger (S5-S6) ECL per domain make up the limited extracellular surface of the channel accessible to biologicals (cytoplasmic membrane is marked by dotted lines in top right panel in FIG. 1). The different domains are connected by ICLs (S6-S1) and both N- and C-terminal ends reside at the cytoplasmic side of the channel (marked respectively by N and C in bottom panel FIG. 1). Each domain consists of a voltage sensor domain (VSD; S1-S4) and ion-conducting pore domain (PD; S5-S6) arranged such that the VSD of each domain is closest to the PD of the following domain, in a clockwise orientation. The central Na⁺-conducting pore of the channel (marked by a star in bottom panel 1) is formed by the PDs and their ECLs that line the cavity. FIG. 32 is a schematic representation of Nav1.7α.

Voltage-gated sodium channels may interact with different Navβ-subunits (Navβ1 to Navβ4) that among other things can modulate the channels' electrophysiological properties and cell surface expression levels (reviewed by Winters & Isom 2016 Current Topics in Membranes 78: 315). The bottom panel of FIG. 1 depicts suggested interaction sites for three different Navβ-subunits, according to recent findings (Das et al. 2016 eLIFE 5:e10960; Zhu et al. 2017 J Gen Physiol 149: 813; Yan et al. 2017 Cell 170: 470).

A detailed sequence comparison of the different ECLs of huNav1.7α to their ortholog and paralog counterparts can be found in FIGS. 2A-2B. Different splice variants of Nav1.7α exist that through interaction with Navβ1 impact on the electrophysiological properties of the channel (Chatelier et al. 2008 J Neurophysiol 99: 2241; Farmer et al. 2012 PLoS ONE 7: e41750). The major technical drawbacks of Nav1.7α as a target for biologicals are its poor cell surface expression level combined with a limited accessibility to the extracellular surface.

BRIEF SUMMARY OF THE INVENTION

The present invention provides Nav1.7 binders, which are immunoglobulin single variable domains (ISVDs) that bind and inhibit Nav1.7α channels with exquisite selectivity over other Nav channel paralogs. The Nav1.7 binders may be useful for preparing formulations for treating chronic pain or pain.

The present invention provides Nav1.7 binders that bind to a human voltage-gated sodium channel Nav1.7α protein subunit (human NaV1.7a subunit) between amino acids 272 and 331 of the human NaV1.7α subunit Domain 1 S5-S6 loop, wherein the human NaV1.7α subunit comprises the amino acid sequence set forth in SEQ ID NO: 1. In particular embodiments, the Nav1.7 binder contacts amino acids F276, R277, E281, and V331 of the human NaV1.7α subunit, which in particular embodiments, binds to the human NaV1.7α subunit with lower affinity than to human NaV1.7α subunit lacking such substitutions. In certain embodiments, the Nav1.7 binder further is capable of binding a rhesus monkey human NaV1.7α subunit with a lower affinity than it binds to the human NaV1.7α subunit.

The Nav1.7 binder is an antibody or an antibody fragment, which in specific embodiments is a heavy chain antibody or an ISVD. In particular embodiments, the heavy chain antibody is a camelid antibody and the ISVD is a VHH.

In particular embodiments, the Nav1.7 binder comprises (a) a complementarity determining region (CDR) 1, CDR1, comprising the amino acid sequence set forth in SEQ ID NO: 247, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 248, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 249; or (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 250, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 251, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 252; or (c) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254, and a CDR3 comprising the amino acid sequence SRY; or (d) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 256, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 257, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 258; or (e) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 259, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 260, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 261; or (f) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 262, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 263, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 264; or (g) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; or (h) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206; or (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219; or (j) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 221, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225.

In a further embodiment, the Nav1.7 binder comprises (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196 or SEQ ID NO: 197; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 198 or SEQ ID NO: 199; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; or (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, or SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206; or (c) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, or SEQ ID NO: 212; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, or SEQ ID NO: 218; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219; or (d) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201 or SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223 or SEQ ID NO: 224; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 233; or (e) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206; or (0 a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219; or (g) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 233.

In a further embodiment the Nav1.7 binder comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; or (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; or (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, and SEQ ID NO: 153; or (d) an amino acid sequence selected from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.

In particular embodiments, the Nav1.7 binder comprises a C-terminal alanine residue.

In particular embodiments, the Nav1.7 binder is conjugated to a half-life extender, which in certain embodiments is a human serum albumin (HSA) binder or the crystallizable fragment (Fc) of an antibody. HSA binders include but are not limited ALB11002 or ALB00223. In particular embodiments, the Nav1.7 binder is conjugated to is polyethylene glycol, which provides half-life extension.

The present invention further provides for use of a Nav1.7 binder disclosed herein for the manufacture of a medicament for the treatment of chronic pain.

The present invention further provides for use of a Nav1.7 binder disclosed herein for the treatment of chronic pain.

The present invention further provides a method for treating an individual with chronic pain comprising administering to the individual a therapeutically effective amount of a Nav1.7 binder disclosed herein to treat the chronic pain. The individual may be a human patient in need of pain relief. The human patient may be treated in a hospital setting or in an out-patient setting. The Nav1.7 binder may be administered by syringe, autoinjector, dose-settable delivery device, or the like.

The present invention further provides a composition comprising a Nav1.7 binder disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides a nucleic acid molecule encoding the Nav1.7 binder disclosed herein. In a further embodiment the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 273-283. In a further embodiment the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 284-421.

The present invention further provides a vector comprising the nucleic acid molecule encoding a Nav.7 binder. The present invention further provides a host cell comprising a nucleic acid molecule encoding a Nav1.7 binder disclosed herein.

The present invention further provides a method for producing a Nav1.7 binder disclosed herein comprising: (a) providing a host cell comprising a nucleic acid molecule encoding a Nav1.7 binder disclosed herein or a vector comprising a nucleic acid molecule encoding the Nav1.7 binder disclosed herein; (b) cultivating the host cell in a medium under conditions suitable for expression of the Nav1.7 binder by the host cell; and (c) isolating the Nav1.7 binder from the medium to provide the Nav1.7 binder.

The present invention further provides a Navβ1 binder comprising (a) a first immunoglobulin single variable domain (ISVD) comprising three complementarity determining regions (CDRs) wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 425, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 426, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 427; or (b) a second ISVD comprising three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 437, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 438, and CDR3 comprise the amino acid sequence set forth in SEQ ID NO: 439.

Ina further embodiment of the Navβ1 binder, the first ISVD comprises the amino acid sequence set forth in SEQ ID NO: 411 and the second ISVD comprises the amino acid sequence set forth in SEQ ID NO: 415. In a further embodiment, the N-terminal amino acid of the first ISVD or the second ISVD is linked to the C-terminal amino acid of a Nav1.7 binder of claim 1 by a peptide or polypeptide linker or the N-terminal amino acid of the Nav1.7 binder of claim 1 is linked to the C-terminal amino acid of the first ISVD or the second ISVD by a peptide or polypeptide linker.

In further embodiments of the Navβ1 binder, the peptide or polypeptide linker comprises any combination of glycine and serine amino acids up to 40 amino acids. In a further embodiment of the Navβ1 binder, the peptide or polypeptide linker comprises an amino acid sequence comprising GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In a particular embodiment, the polypeptide linker comprises the amino acid sequence set forth in SEQ ID NO: 463.

The present invention further provides a nucleic acid molecule encoding a Navβ1 binder disclosed herein. In a further embodiment, the Navβ1 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 456 and 461.

The present invention further provides a vector comprising the nucleic acid molecule encoding a Navβ1 binder disclosed herein. The present invention further provides a host cell comprising a nucleic acid molecule encoding a Navβ1 binder disclosed herein.

The present invention further provides a method for producing a Navβ1 binder disclosed herein comprising: (a) providing a host cell comprising a nucleic acid molecule encoding a Navβ1 binder disclosed herein or a vector comprising a nucleic acid molecule encoding the Navβ1 binder disclosed herein; (b) cultivating the host cell in a medium under conditions suitable for expression of the Navβ1 binder by the host cell; and (c) isolating the Navβ1 binder from the medium to provide the Navβ1 binder.

The present invention further provides a Navβ2 binder comprising (a) a first immunoglobulin single variable domain (ISVD) comprising three complementarity determining regions (CDRs) wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 422, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 423, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 424; (b) a second ISVD comprising three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 428, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 429, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 430; (c) a third ISVD comprising three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 431, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 432, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 433; or (d) a fourth ISVD comprising three CDRs wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 434, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 435, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 436.

In a further embodiment of the Navβ2 binder, the first ISVD comprises the amino acid sequence set forth in SEQ ID NO: 410, the second ISVD comprises the amino acid sequence set forth in SEQ ID NO: 412, the third ISVD comprises the amino acid sequence set forth in SEQ ID NO: 413, and the fourth ISVD comprises the amino acid sequence set forth in SEQ ID NO: 414.

In a further embodiment of the Navβ2 binder, the N-terminal amino acid of the first ISVD, the second ISVD, the third ISVD, or the fourth ISVD is linked to the C-terminal amino acid of a Nav1.7 binder of claim 1 by a peptide or polypeptide linker or the N-terminal amino acid of the Nav1.7 binder of claim 1 is linked to the C-terminal amino acid of the first ISVD, the second ISVD, the third ISVD, or the fourth ISVD by a peptide or polypeptide linker.

In a further embodiment, the peptide or polypeptide linker comprises any combination of glycine and serine amino acids up to 40 amino acids. In further embodiments, the peptide or polypeptide linker comprises an amino acid sequence comprising GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10. In particular embodiments, the polypeptide linker comprises the amino acid sequence set forth in SEQ ID NO: 463.

The present invention further provides a nucleic acid molecule encoding a Navβ2 binder disclosed herein. In a further embodiment, the Navβ1 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 456, 458, 459, and 460.

The present invention further provides a Nav1.7-Navβ bispecific binder comprising a Nav1.7 binder as disclosed herein and a Navβ binder selected from the group consisting of the Navβ1 binder or Navβ2 binder as disclosed herein.

In further embodiments of the Nav1.7-Navβ bispecific binder, (a) the Nav1.7 binder comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55; (ii) an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; or (iii) an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; or (iv) an amino acid sequence selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, and SEQ ID NO: 153; or (v) an amino acid sequence selected from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195; (b) the Navβ1 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 411 and SEQ ID NO: 415; and, (c) the Navβ2 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 410, SEQ ID NO: 412, SEQ ID NO: 413, and SEQ ID NO: 414.

The present invention further provides a Nav1.7-Navβ bispecific binder wherein the Nav1.7-Navβ bispecific binder is linked to a half-life extender.

The present invention further provides a Nav1.7-Navβ bispecific binder disclosed herein wherein the half-life extender is a human serum albumin (HSA) binder or HC constant domain or crystallizable fragment (Fc domain). The present invention further provides a Nav1.7-Navβ bispecific binder disclosed herein wherein the Nav1.7-Navβ bispecific binder comprises a C-terminal alanine residue.

The present invention further provides a composition comprising a Nav1.7-Navβ bispecific binder disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides for the use of a Nav1.7-Navβ bispecific binder disclosed herein for the manufacture of a medicament for the treatment of chronic pain.

The present invention further provides a Nav1.7-Navβ bispecific binder disclosed herein or a composition comprising said Nav1.7-Navβ bispecific binder for the treatment of chronic pain.

The present invention further provides a method for treating an individual with chronic pain comprising administering to the individual a therapeutically effective amount of the Nav1.7-Navβ bispecific binder disclosed herein or a composition comprising said Nav1.7-Navβ bispecific binder to treat the chronic pain.

The present invention further provides a nucleic acid molecule encoding a Nav1.7-Navβ bispecific binder comprising a nucleic acid molecule encoding a Nav1.7 binder disclosed herein and a Navβ1 or Navβ2 binder disclosed herein. In a further embodiment, the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 273-283, the Navβ1 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 457 and 461, and Navβ2 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 456, 458, 459, and 460. In a further embodiment, the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 284-421, the Navβ1 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 457 and 461, and Navβ2 binder comprises a nucleotide sequence selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 456, 458, 459, and 460.

The present invention further provides a vector comprising the nucleic acid molecule encoding a Nav1.7-Navβ bispecific binder disclosed herein. The present invention further provides a host cell comprising a nucleic acid molecule encoding a Nav1.7-Navβ bispecific binder disclosed herein.

The present invention further provides a method for producing a Nav1.7-Navβ bispecific binder disclosed herein comprising: (a) providing a host cell comprising a nucleic acid molecule encoding a Nav1.7-Navβ bispecific binder disclosed herein or a vector comprising a nucleic acid molecule encoding the Nav1.7-Navβ bispecific binder disclosed herein; (b) cultivating the host cell in a medium under conditions suitable for expression of the Nav1.7-Navβ bispecific binder by the host cell; and (c) isolating the Nav1.7-Navβ bispecific binder from the medium to provide the Nav1.7-Navβ bispecific binder.

The present invention further provides a Nav1.7 binder, Navβ1 binder, or Navβ2 binder comprising an amino acid sequence disclosed in Table 56. The present invention further provides a nucleic acid molecule encoding a Nav1.7 binder, Navβ1 binder, or Navβ2 binder and comprising a nucleotide sequence having at least 80, 90%, 95%, or 100% identity to a nucleotide sequence disclosed in Table 56 provided the amino acid sequence encoded by the nucleotide sequence is disclosed in Table 56. The present invention further provides a Nav1.7-Navβ bispecific binder comprising an amino acid sequence disclosed in Table 56 or comprised of a Nav1.7 binder and at least one Navβ binder selected from Navβ1 binder and Navβ2 binder, each comprising an amino acid sequence disclosed in Table 56. The present invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding a Nav1.7-Navβ bispecific binder wherein the nucleotide sequence has at least 80, 90%, 95%, or 100% identity to a nucleotide sequence disclosed in Table 56 provided the nucleotide sequence encodes an amino acid sequence disclosed in Table 56.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the proposed structure of Nav1.7α. Drawing shows a huNav1.7α model viewed from top/extracellular (top left panel) and side through cytoplasmic membrane (top right panel). Nav1.7α structural topology viewed from extracellular side (bottom panel) shown with β1, β2, and β3 subunits.

FIG. 2A and FIG. 2B together show sequence comparisons of huNav1.7α to paralogs and orthologs (based on sequences listed in the Table 41).

FIG. 3A shows the binding of ISVDs F103262CO2, F0103265B04, F0103262B06, F0103265A11 to huNav1.7α+β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3B shows the binding of ISVD F0103362B08 to huNav1.7α++β1−β2−β3. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

FIG. 3C shows the binding of ISVDs F103262CO2, F0103265B04, F0103262B06, F0103265A11 to huNav1.7α+β1. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3D shows the binding of ISVDs F103262CO2, F0103265B04, F0103262B06, F0103265A11 to huNav1.5α−β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3E shows the binding of ISVDs F0103265B04 and F0103262B08, to huNav1.7+β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3F shows the binding of ISVDs F0103265B04 and F0103262B08, to huNav1.5α−β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3G shows the binding of ISVDs F103262CO2, F0103265B04, F0103262B06, F0103265A11 to huNav157chimera14−β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3H shows the binding of ISVDs F0103265B04 and F0103262B08, to huNav1.7α+β1. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 3I shows the binding of ISVDs F0103265B04 and F0103262B08, to huNav157chimera14−β1−β2−β3. MFI=median fluorescence intensity; IRR=irrelevant control ISVD; a-FLAG is a detection moiety.

FIG. 4 shows a sequence alignment of functional Nav1.7α+selective ISVDs compared to the human VH3-JH consensus sequence (SEQ ID NO: 57). Residues identical to the human VH3-JH consensus are shown by dots. CDRs are highlighted. The amino acid sequences for the ISVDs are F0103265B04 (SEQ ID NO: 49); F0103275B05 (SEQ ID NO: 50), F0103387G04 (SEQ ID NO: 52); F0103265A11 (SEQ ID NO: 48); F0103387G05 (SEQ ID NO: 53); F0103362B08 (SEQ ID NO: 51).

FIG. 5 shows screening of the F0103275B05 (275B05) stage I affinity maturation library in binding fluorescence-activated cell sorting (FACS) on huNav1.7α and rhNav1.7α.

FIG. 6 shows screening of the F0103275B05 (275B05) stage II affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 7A shows a schematic for a single pulse electrophysiology protocol.

FIG. 7B shows a schematic for a two pulse electrophysiology protocol.

FIG. 8 shows screening of the F0103265A11 (265A11) stage I affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 9 shows screening of the F0103265A11 (265A11) stage II affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 10 shows screening of the F0103265B04 (265B04) stage I affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 11 shows screening of the F0103387G05 (387G05) stage I affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 12 shows screening of the F0103362B08 (362B08) stage I affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 13 shows screening of the F0103464B09 (464B09) stage I affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 14 shows screening of the F0103464B09 (464B09) stage II affinity maturation library in binding FACS on huNav1.7α and rhNav1.7α.

FIG. 15A shows competition FACS of extracellular anti-Nav1.7α ISVDs vs. F0103265B04 on stable HEK cell lines expressing huNav1.7+β1−β2−β3.

FIG. 15B shows competition FACS of extracellular anti-Nav1.7α ISVDs vs. F0103265B04 on stable HEK cell lines expressing huNav1.7α+β1−β2−β3.

FIG. 15C shows competition FACS of extracellular anti-Nav1.7α ISVDs vs. F0103275B05(N93R) on stable CHO cell lines expressing huNav1.7α+β1−β2−β3.

FIG. 15D shows competition FACS of extracellular anti-Nav1.7α ISVDs vs. F0103275B05(N93R) on stable CHO cell lines expressing rhNav1.7α+β1−β2−β3.

FIG. 16 shows a schematic overview of huNav1.7α+huNav1.5α (huNav157) chimeras.

FIG. 17A, FIG. 17B, and FIG. 17C together show epitope mapping FACS of extracellular anti-Nav1.7α ISVDs (1 μM) on transiently transfected cells expressing huNav157+β1−β2−β3 chimeras 1, 2, 3, or 4 (huNav157chim1, huNav157chim2, huNav157chim3, or huNav157chim4, respectively) compared to cells expressing huNav1.7α+β1−β2−β3.

FIG. 18A, FIG. 18B, and FIG. 18C together show epitope mapping FACS of extracellular anti-Nav1.7α ISVDs (1 μM) on transiently transfected cells expressing huNav157+β1−β2−β3 chimeras 5, 6, 7, or 8 (huNav157chim5, huNav157chim6, huNav157chim7, or huNav157chim8, respectively) compared to cells expressing huNav1.7α+β1−β2−β3.

FIG. 19A and FIG. 19B together show epitope mapping FACS of extracellular anti-Nav1.7α ISVDs (1 μM) on transiently transfected cells expressing huNav157+β1−β2−β3 chimeras 9 or 12 (huNav157chim9 or huNav157chim12, respectively) compared to cells expressing huNav1.7α+β1−β2−β3.

FIG. 20A and FIG. 20B together show epitope mapping FACS of extracellular anti-Nav1.7α ISVDs (1 μM) on transiently transfected cells expressing huNav157+β1−β2−β3 chimeras 22 or 18 (huNav157chim22 or huNav157chim18, respectively) compared to cells expressing huNav1.7α+β1−β2−β3.

FIG. 21A and FIG. 21B together show shows epitope mapping FACS of extracellular anti-Nav1.7α ISVDs (1 μM) on transiently transfected cells expressing huNav1.7+β1−β2−β3, rhNav1.7+β1−β2−β3 or huNav1.7(N146S, V1941, F276V, R277Q, E281V, V331M, E504D, D507E, S508N, N533S)−β1−β2−β3.

FIG. 22A shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn huNav1.7α+β1−β2−β3.

FIG. 22B shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn RhNav1.7α+β1−β2−β3.

FIG. 22C shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn Nav1.7α(F276V)+β1−β2−β3.

FIG. 22D shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn Nav1.7α(R277Q)+β1−β2−β3.

FIG. 22E shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn Nav1.7α(E281V)+β1−β2−β3.

FIG. 22F shows binding FACS of extracellular anti-Nav1.7α ISVDs on stable huNav1.7α-rhNav1.7α chimera cell line CHO FlpIn Nav1.7α(V331M)+β1−β2−β3.

FIG. 22G shows a schematic representation of the extracellular polymorphisms between huNav1.7α and rhNav1.7α on an huNav1.7α model viewed from the extracellular side.

FIG. 23A shows a schematic illustrating the IonFlux 16 single pulse protocol.

FIG. 23B shows a schematic illustrating the IonFlux 16 two pulse protocol.

FIG. 24A shows an IonFlux 16 dose response titration of F0103265B04, F0103362B08, F0103387G04 and F0103387G05 using the single pulse (P1) protocol.

FIG. 24B shows an IonFlux 16 dose response titration of F0103265B04, F0103362B08, F0103387G04 and F0103387G05 using two pulse (P2) protocol.

FIG. 25A shows an IonFlux 16 single high concentration dose response for F0103265B04, F0103275B05, and F0103262CO2 in HEK huNav1.7α+β1 cells using single pulse (P1) and two pulse (P2) protocols.

FIG. 25B shows an IonFlux 16 single high concentration dose response for F0103265B04, F0103275B05, and F0103262CO2 in HEK huNav1.7α cells using single pulse (P1) and two pulse (P2) protocols.

FIG. 25C shows an IonFlux 16 single high concentration dose response for F0103265B04, F0103275B05, and F0103262CO2 in CHO FlpIn huNav1.7α+β1−β2−β3 cells using single pulse (P1) and two pulse (P2) protocols.

FIG. 25D shows an IonFlux 16 single high concentration dose response for F0103262B06, F0103265A11, and F0103265B04 in CHO FlpIn huNav1.7α+β1−β2−β3 cells using single pulse (P1) and two pulse (P2) protocols.

FIG. 25E shows an IonFlux 16 single high concentration dose response for F0103262B06, F0103265A11, and F0103265B04 in HEK FlpIn huNav1.7α+β1−β2−β3 cells using single pulse (P1) and two pulse (P2) protocols.

FIG. 26 shows the results of an IonFlux 16 washout experiment using F0103265B04.

FIG. 27 shows the results of an IonFlux 16 time course experiment using F0103265B04.

FIG. 28 shows a sequence analysis of F0103275B05 (SEQ ID NO: 50) and F010387G04 (SEQ ID NO: 52) compared to the human VH3-JH consensus sequence (SEQ ID NO: 57), VHH2 consensus sequence (SEQ ID NO: 58), and sequenced optimized F0103387G04 (F0103387G04 SO; SEQ ID NO:59).

FIG. 29 shows a sequence analysis of F0103387G05 (SEQ ID NO: 53) compared to the human VH3-JH consensus sequence (SEQ ID NO: 57), VHH2 consensus sequence (SEQ ID NO: 58), and sequenced optimized F0103387G05 (F0103387G05_SO; SEQ ID NO:60).

FIG. 30 shows the Tm of F0103387G05 variants in function of pH. Dotted lines mark variants with H37Y substitution (see Table 30).

FIG. 31 shows a sequence analysis of F0103464B09 (SEQ ID NO: 55) compared to the human VH3-JH consensus sequence (SEQ ID NO: 57), VHH2 consensus sequence (SEQ ID NO: 58), and sequenced optimized F01034647B09 (F01034647B09_SO; SEQ ID NO:61).

FIG. 32 shows a schematic diagram of huNav1.7α. VSD=voltage sensing domain; PM=pore module; D=domain; S=transmembrane segment.

FIG. 33 shows results of a binding FACS of anti-Navβ2 ISVD F0103240B04 on stable cell lines.

FIG. 34A shows results of a binding ELISA of the shown anti-Navβ ISVDs binding to Navβ1. F0103240B04 is a potent anti-Navβ2 binder control and IRR022 is a negative control comprising an irrelevant binder. F0103478E09 weakly binds Navβ1.

FIG. 34B shows results of a binding ELISA of the shown anti-Navβ ISVDs binding to 132. F0103240B04 is a potent anti-Navβ2 binder control and IRR0022 is a negative control comprising an irrelevant binder. F0103492E09, F0103500E03, and F0103505D08 weakly bind 132.

FIG. 34C shows results of a binding ELISA of the shown anti-Navβ ISVDs binding to Navβ3. F0103240B04 is a potent anti-Navβ2 binder control and IRR0202 is a negative control comprising an irrelevant binder. None of the ISVDs bind Navβ3.

FIG. 35A, FIG. 35B, FIG. 35C, and FIG. 35D together show results of binding FACS of the shown anti-Navβ subunit ISVDs (12.3 nM) on transiently transfected cells. Positive controls anti-Navβ1, anti-Navβ2, and anti-Navβ3 are rabbit polyclonal antibodies specific for human Navβ1, Navβ2, and Navβ3, respectively.

FIG. 36A shows results of binding FACS of anti-Navβ ISVD F0103478E09 on various stable cell lines.

FIG. 36B shows results of binding FACS of anti-Navβ ISVD F0103492E09 on various stable cell lines.

FIG. 36C shows results of binding FACS of anti-Navβ ISVD F0103500E03 on various stable cell lines.

FIG. 36D shows results of binding FACS of anti-Navβ ISVD F0103505D08 on various stable cell lines.

FIG. 36E shows results of binding FACS of anti-Navβ ISVD F0103495D09 on various stable cell lines.

FIG. 37A shows the results of a competition FACS of Nav1.7α-Navβ bispecific ISVDs on stable CHO cell lines expressing human Nav1.7α-Navβ1-Navβ2-Navβ3 (Nav1.7-β1-β2-β3).

FIG. 37B shows the results of a competition FACS of Nav1.7α-Navβ bispecific ISVDs on stable CHO cell lines expressing rhesus Nav1.7α-Navβ1-Navβ2-Navβ3 (Nav1.7-β1-β2-β3).

FIG. 38A shows the results of a competition FACS of Nav1.7α-Navβ bispecific ISVDs on stable HEK cell lines expressing human Nav1.7α (Nav1.7).

FIG. 38B shows the results of a competition FACS of Nav1.7α-Navβ bispecific ISVDs on stable HEK cell lines human expressing Nav1.7α-Navβ1 (Nav1.7-β1).

FIG. 38C shows the results of a competition FACS of Nav1.7α-Navβ bispecific ISVDs on stable HEK cell lines expressing human Nav1.7α-Navβ1-Navβ2-Navβ3 (Nav1.7-β1-β2-β3).

FIG. 39A shows binding FACS of Nav1.7 binder F0103262C02 on stable huNav1.x paralog HEK293T cell lines. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

FIG. 39B shows binding FACS of Nav1.7 binder F0103265B04 on stable huNav1.x paralog HEK293T cell lines. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

FIG. 39C shows binding FACS of Nav1.7 binder F0103275B05 on stable huNav1.x paralog HEK293T cell lines. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

FIG. 39D shows binding FACS of Nav1.7 binder F0103464B09 on stable huNav1.x paralog HEK293T cell lines. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

FIG. 39E shows binding FACS of Nav1.7 binder F0103387G05 on stable huNav1.x paralog HEK293T cell lines. MFI=median fluorescence intensity; a-FLAG is a detection moiety.

DETAILED DESCRIPTION OF THE INVENTION
Definitions

As used herein, the term “Nav1.7 binder” refers to an antibody, an antibody fragment, an immunoglobulin single variable domain (also referred to as “ISV” or ISVD″) or single domain antibody (also referred to as “sdAb”) that binds to Nav1.7α. An example of an ISVD is a Nanobody® molecule.

As used herein, the term “Navβ binder” refers to an antibody, an antibody fragment, an immunoglobulin single variable domain (also referred to as “ISV” or ISVD″) or single domain antibody (also referred to as “sdAb”) that binds to Navβ. The term “Navβ” comprises the terms “Navβ1” and “Navβ2”.

As used herein, “antibody” refers to an entire immunoglobulin, including recombinantly produced forms and includes any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, biparatopic antibodies, and chimeric antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as humanization of a non-human antibody for use as a human therapeutic antibody.

The term “antibody” refers, in one embodiment, to a conventional antibody, which is a protein tetramer comprising two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds, or an antigen binding portion thereof, and in another embodiment, to a nonconventional antibody, which is a heavy chain antibody protein dimer comprising two heavy chains inter-connected by disulfide bonds and no light chains, or antigen binding portion thereof. In either embodiment, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as V_H) and a heavy chain constant region or domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, C_H1, C_H2 and C_H3. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region or domain (abbreviated herein as V_L) and a light chain constant region or domain. The light chain constant region is comprised of one domain, CL. The human V_Hincludes six family members: V_H1, V_H2, V_H3, V_H4, V_H5, and V_H6 and the human V_Lfamily includes 16 family members: V_κ1, V_κ2, V_κ3, V_κ4, V_κ5, V_κ6, V_λ1, V_λ2, V_λ3, V_λ4, V_λ5, V_λ6, V_λ7, V_λ8, V_λ9, and V_λ10. Each of these family members can be further divided into particular subtypes.

The V_Hand V_Lregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_Hand V_Lis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs form a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

The constant domains or regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgG₁(Eu), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgG₁described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgG₁, IgG₂, IgG₃, and IgG₄constant domains in Beranger, et al., Ibid.

The variable domains or regions of the heavy and light chains contain a binding domain comprising the CDRs that interacts with an antigen. A number of methods are available in the art for defining or predicting the CDR amino acid sequences of antibody variable domains (see Dondelinger et al., Frontiers in Immunol. 9: Article 2278 (2018)). The common numbering schemes include the following.

- Kabat numbering scheme is based on sequence variability and is the most commonly used (See Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence); Chothia numbering scheme is based on the location of the structural loop region (See
- Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997));
- AbM numbering scheme is a compromise between the two used by Oxford Molecular's
- AbM antibody modelling software (see Karu et al, ILAR Journal 37: 132-141 (1995);
- Contact numbering scheme is based on an analysis of the available complex crystal structures (See www.bioinf.org.uk: Prof Andrew C. R. Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008).
- IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all the protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains as well as T cell receptor chains from different species and counts residues continuously from 1 to 128 based on the germ-line V sequence alignment (see Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509(1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003)).
  
  While there are several different methods for determining the amino acid sequences of the CDRs, the numbering of the entire variable region typically follows the Kabat numbering scheme with the particular CDR numbering scheme imposed thereupon.

The following general rules disclosed in www.bioinforg.uk: Prof. Andrew C. R. Martin's Group and reproduced in Table 1 below may be used to define or predict the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.

TABLE 1

Loop
Kabat
AbM
Chothia¹
Contact²
IMGT

L1
L24--L34
L24--L34
L24--L34
L30--L36
L27--L32

L2
L50--L56
L50--L56
L50--L56
L46--L55
L50--L52

L3
L89--L97
L89--L97
L89--L97
L89--L96
L89--L97

H1
H31--H35B
H26--H35B
H26--H32 . . . 34
H30--H35B
H26--H35B

(Kabat Numbering)³

H1
H31--H35
H26--H35
H26--H32
H30--H35
H26--H33

(Chothia Numbering)

H2
H50--H65
H50--H58
H52--H56
H47--H58
H51--H56

H3
H95--H102
H95--H102
H95--H102
H93--H101
H93--H102

¹Some of these numbering schemes (particularly for Chothia loops) vary depending on the individual publication examined.

²Any of the numbering schemes can be used for these CDR definitions, except the Contact numbering scheme uses the Chothia or Martin (Enhanced Chothia) definition.

³The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme places the insertions at H35A and H35B.)

If neither H35A nor H35B is present, the loop ends at H32

If only H35A is present, the loop ends at H33

If both H35A and H35B are present, the loop ends at H34

In general, the state of the art recognizes that in many cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on CDR3 of the heavy chain alone are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910⁻⁸⁹¹⁵(1998); Xu et al., Immunity 13: 37-45 (2000).

A conventional antibody tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The heavy chain of a conventional antibody may or may not contain a terminal lysine (K), or a terminal glycine and lysine (GK).

As used herein, “antigen binding fragment” or “antigen binding portion” refers to fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; single-chain antibody molecules, e.g., sc-Fv; immunoglobulin single variable domain molecules, and multispecific antibodies formed from antibody fragments.

As used herein, the term “immunoglobulin single variable domain” (also referred to as “ISV” or ISVD″) or “single domain antibody (also referred to as “sdAb”) are terms that are used to refer to immunoglobulin variable domains (which may be heavy chain or light chain domains, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interaction with another variable domain (e.g., without a VH/VL interaction as is required between the VH and VL domains of a conventional four-chain monoclonal antibody). The term “VH” refers to a heavy chain variable domain of a conventional antibody and the term “VHH” refers to the heavy chain variable domain of a non-conventional heavy chain antibody.

Examples of ISVDs include for example, VHHs, humanized VHHs, and/or a camelized VHs such as camelized human VHs), IgNAR domains, single domain antibodies such as dAbs™, which are VH domains or are derived from a VH domain or are VL domains or are derived from a VL domain. ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, an ISVD will be a VHH, a humanized VHH, or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (e.g., optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression).

The term “Nanobody® molecule” is generally as defined in WO 2008/020079 or WO 2009/138519, and thus in a specific aspect denotes an VHH, a humanized VHH, or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as, e.g., optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression). The term Nanobody® is a registered trademark of Ablynx N.V.

As used herein, “Nav1.7 binder” refers to a conventional antibody, heavy chain antibody, antigen binding fragment of an antibody or ISVD that binds to Nav1.7α. A Nav1.7 binder may be part of a larger molecule such as a multivalent, bispecific, or multispecific binder that includes one or more Nav1.7 binders and may include one or more binders to a target other than Nav1.7α (e.g., Navβ binder) and may comprises another functional element, such as, for example, a half-life extender (HLE), an Fc domain of an immunoglobulin, a targeting unit and/or a small molecule such a polyethylene glycol (PEG).

As used herein, “Navβ binder” refers to a conventional antibody, heavy chain antibody, antigen binding fragment of an antibody or ISVD that binds to Navβ1 or Navβ2. A Navβ binder may be part of a larger molecule such as a multivalent, bispecific, or multispecific binder that includes one or more Navβ binders and may include one or more binders to a target other than Navβ1 or Navβ2 (e.g., a Nav1.7 binder) and may comprise another functional element, such as, for example, a half-life extender (HLE), an Fc domain of an immunoglobulin, a targeting unit and/or a small molecule such as a PEG. Monovalent, monospecific and/or biparatopic Nav1.7 or Navβ binders are part of the present invention. A monovalent Nav1.7 or Navβ binder (e.g., ISVD such as a Nanobody® molecule) is a molecule that comprises a single antigen-binding domain. A bivalent or bispecific Nav1.7 binder (e.g., ISVD such as a Nanobody® molecule) comprises two antigen-binding domains, e.g., a Nav1.7-Navβ bispecific binder. A multivalent or multispecific Nav1.7 binder comprises more than one antigen-binding domain (e.g., 1, 2, 3, 4, 5, 6, or 7). When a multivalent or multispecific binder comprises only two antigen binding domains it may be referred to as a bispecific or bivalent binder.

For a general description of multivalent and multispecific polypeptides containing one or more ISVDs and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 1996/34103, WO 1999/23221, WO 2004/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

As used herein, a “Fab fragment” is comprised of one light chain and the C_H1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.

As used herein, a “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the C_H1 domain and also the region between the C_H1 and C_H2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′)₂molecule.

As used herein, a “F(ab′)₂fragment” contains two light chains and two heavy chains containing the V_Hdomain and a portion of the constant region between the C_H1 and C_H2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab′)₂fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab′)₂fragment” can be the product of pepsin cleavage of an antibody.

As used herein, an “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

As used herein, an “Fc domain” or “Fc region” each refer to the fragment crystallizable region of an antibody. The Fc domain comprises two heavy chain fragments comprising the C_H1 and C_H2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_H3 domains. The Fc domain may be fused at the N-terminus or the C-terminus to a heterologous protein.

As used herein, a “diabody” refers to a small antibody fragment with two antigen-binding regions, which fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H-V_Lor V_L-V_H). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding regions. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, “isolated” antibodies or antigen-binding fragments thereof (e.g., Nav1.7 and Navβ binders) are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

As used herein, a “monoclonal antibody” refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991)J Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

As used herein, a “humanized ISVD” or “humanized antibody” refers to forms of Nav1.7 binders that contain sequences from both human and non-human (e.g., llama, murine, rat) antibodies. In general, the humanized Nav1.7 and Navβ binders will comprise all of at least one, and typically two, variable domains, in which the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized Nav1.7 and/or Navβ binder may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

“Humanization” (also called Reshaping or CDR-grafting) is now a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent or camelids) and for improving the effector functions (ADCC, complement activation, C1q binding). The engineered mAb is engineered using the techniques of molecular biology, however simple CDR-grafting of the rodent complementarity-determining regions (CDRs) into human frameworks often results in loss of binding affinity and/or specificity of the original mAb. In order to humanize an antibody, the design of the humanized antibody includes variations such as conservative amino acid substitutions in residues of the CDRs, and back substitution of residues from the rodent mAb into the human framework regions (backmutations). The positions can be discerned or identified by sequence comparison for structural analysis or by analysis of a homology model of the variable regions' 3D structure. The process of affinity maturation has most recently used phage libraries to vary the amino acids at chosen positions. Similarly, many approaches have been used to choose the most appropriate human frameworks in which to graft the rodent CDRs. As the datasets of known parameters for antibody structures increases, so does the sophistication and refinement of these techniques. Consensus or germline sequences from a single antibody or fragments of the framework sequences within each light or heavy chain variable region from several different human mAbs can be used. Another approach to humanization is to modify only surface residues of the rodent sequence with the most common residues found in human mAbs and has been termed “resurfacing” or “veneering.” Known human Ig sequences are disclosed, e.g.,

- www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;
- www.atcc.org/phage/hdb.html; www.kabatdatabase.com/top.html;
- www.antibodyresource.com/onlinecomp.html; www.appliedbiosystems.com;
- www.biodesign.com; antibody.bath.ac.uk; www.unizh.ch; www.cryst.bbk.ac.uk/.about.ubcgO7s; Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), each entirely incorporated herein by reference. Often, the human or humanized antibody is substantially non-immunogenic in humans.

As used herein, “non-human amino acid sequences” with respect to antibodies or immunoglobulins refers to an amino acid sequence that is characteristic of the amino acid sequence of a non-human mammal. The term does not include amino acid sequences of antibodies or immunoglobulins obtained from a fully human antibody library where diversity in the library is generated in silico (See for example, U.S. Pat. No. 8,877,688 or 8,691,730).

As used herein, “effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

As used herein, “conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in the table below.

Original residue
Conservative substitution

Ala (A)
Gly; Ser

Arg (R)
Lys; His

Asn (N)
Gln; His

Asp (D)
Glu; Asn

Cys (C)
Ser; Ala

Gln (Q)
Asn

Glu (E)
Asp; Gln

Gly (G)
Ala

His (H)
Asn; Gln

Ile (I)
Leu; Val

Leu (L)
Ile; Val

Lys (K)
Arg; His

Met (M)
Leu; Ile; Tyr

Phe (F)
Tyr; Met; Leu

Pro (P)
Ala

Ser (S)
Thr

Thr (T)
Ser

Trp (W)
Tyr; Phe

Tyr (Y)
Trp; Phe

Val (V)
Ile; Leu

As used herein, the term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., Nav1.7α, Navβ1, Navβ2) to which a binder specifically binds. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. A contiguous linear epitope comprises a peptide domain on an antigen comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. A noncontiguous conformational epitope comprises one or more peptide domains or regions on antigen bound by a binder interspersed by one or more amino acids or peptide domains not bound by the binder, each domain independently comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods for determining what epitopes are bound by a given binder (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides (e.g., from Nav1.7α, Navβ1, Navβ2) are tested for reactivity with a given binder. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification of the molecular determinants on the antigen involved in antibody-antigen recognition.

The term “binds to the same epitope” with reference to two or more binders means that the binders bind to the same segment of amino acid residues on a target, as determined by a given method. Techniques for determining whether a particular binder binds to the “same epitope” as the Nav1.7 or Navβ binders described herein include, for example, epitope mapping methods, such as, x-ray analyses of crystals of Nav1.7α:Nav1.7 binder or Navβ:Navβ binder complexes, which provides atomic resolution of the epitope, and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods that monitor the binding of the antibody to antigen fragments (e.g. proteolytic fragments) or to mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component (e.g. alanine scanning mutagenesis—Cunningham & Wells (1985) Science 244:1081). In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the binder of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries.

Binders that “compete with a binder of the present invention for binding to a target antigen” refer to binders that inhibit (partially or completely) the binding of the Nav1.7 binder of the present invention to Nav1.7α or Navβ binder to Navβ. Whether two binders compete with each other for binding to the target antigen, i.e., whether and to what extent one binder inhibits the binding of the other binder to the target antigen, may be determined using known competition experiments. In certain embodiments, a binder competes with, and inhibits binding of a binder of the present invention to the target antigen by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which binder is the “blocking binder” (i.e., the unlabeled binder that is incubated first with the target antigen). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Competing Nav1.7 binders bind to the same epitope as defined herein.

Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, “specifically binds” refers, with respect to a target antigen, to the preferential association of a binder, in whole or part, with the target antigen and not to other molecules, particularly molecules found in human blood or serum. Binders as shown herein typically bind specifically to the target antigen with high affinity, reflected by a dissociation constant (K_D) of 10⁻⁷to 10⁻¹¹M or less. Any K_Dgreater than about 10⁻⁶M is generally considered to indicate nonspecific binding. As used herein, a binder that “specifically binds” or “binds specifically” to a target antigen refers to a binder that binds to the target antigen with high affinity, which means having a K_Dof 10⁻⁷M or less, in particular embodiments a K_Dof 10⁻⁸M or less, or 5×10⁻⁹M or less, or between 10⁻⁸M and 10⁻¹¹M or less, but does not bind with measurable binding to closely related proteins such as human Nav1.1α, human Nav1.2α, human Nav1.3a, humanNav.1.4α, human Nav1.5α, human Nav 1.6α, or human Nav1.8α as determined in a cell ELISA or Surface Plasmon Resonance assay (SPR; Biacore) using 10 μg/mL antibody.

As used herein, an antigen is “substantially identical” to a given antigen if it exhibits a high degree of amino acid sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% or greater amino acid sequence identity to the amino acid sequence of the given antigen. By way of example, an antibody that binds specifically to human Nav1.7α or Navβ may also cross-react with Nav1.7α or Navβ from certain non-human primate species (e.g., rhesus monkey or cynomolgus monkey). The term specifically excludes human Nav1.1α, human Nav1.2α, human Nav1.3a, humanNav.1.4α, human Nav1.5α, human Nav 1.6α, and human Nav1.8a.

As used herein, “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

As used herein, “treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the Nav1.7 and/or Navβ binders of the present invention, topically, subcutaneously, intramuscular, intradermally, or systemically to an individual experiencing chronic pain. The amount of a therapeutic agent that is effective to alleviate chronic pain in the individual may vary according to factors such as the injury or disease state, age, and/or weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual. Whether chronic pain has been alleviated can be assessed by the individual and/or any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of chronic pain. Thus, the terms denote that a beneficial result has been or will be conferred on a human or animal individual experiencing chronic pain.

As used herein, “treatment,” as it applies to a human or veterinary individual, refers to therapeutic treatment, as well as diagnostic applications. “Treatment” as it applies to a human or veterinary individual, encompasses contact of the antibodies or antigen binding fragments of the present invention to a human or animal subject.

As used herein, “therapeutically effective amount” refers to a quantity of a specific substance sufficient to achieve a desired effect in an individual being treated. For instance, this may be the amount necessary to inhibit or reduce the severity of chronic pain in an individual.

As used herein, the term “effector-silent” as used herein refers to an antibody, antibody fragment, HC constant domain, or Fc domain thereof that displays (i) no measurable binding to one or more Fc receptors (FcRs) as may be measured in a surface plasmon resonance (SPR) assay (e.g., Biacore™ assay) wherein an association constant in the micromolar range indicates no measurable binding or (ii) measurable binding to one or more FcRs as may be measured in SPR assay that is reduced compared to the binding that is typical for an antibody, antibody fragment, HC constant domain or Fc domain thereof the same isotype. In particular embodiments, the antibody, antibody fragment, HC constant domain, or Fc domain thereof may comprise one or more mutations in the HC constant domain and the Fc domain in particular such that the mutated an antibody, antibody fragment, HC constant domain or Fc domain thereof has reduced or no measurable binding to FcγRIIIa, FcγRIIa, and FcγRI compared to a wild-type antibody of the same isotype as the mutated antibody. In particular embodiments, the affinity or association constant of an effector-silent an antibody, antibody fragment, HC constant domain or Fc domain thereof to one or more of FcγRIIIa, FcγRIIa, and FcγRI is reduced by at least 1000-fold compared to the affinity of the wild-type isotype; reduced by at least 100-fold to 1000-fold compared to the affinity of the wild-type isotype reduced by at least 50-fold to 100-fold compared to the affinity of the wild-type isotype; or at least 10-fold to 50-fold compared to the affinity of the wild-type isotype. In particular embodiments, the effector-silent an antibody, antibody fragment, HC constant domain, or Fc domain thereof has no detectable or measurable binding to one or more of the FcγRIIIa, FcγRIIa, and FcγRI as compared to binding by the wild-type isotype. In general, effector-silent an antibody, antibody fragment, HC constant domain, or Fc domain thereof will lack measurable antibody-dependent cell-mediated cytotoxicity (ADCC) activity. An ISVD not fused or linked to an effector-silent HC constant domain or Fc domain thereof displays no detectable or measurable binding to one or more of FcγRIIIa, FcγRIIa, or FcγRI. SPR assays measure binding of an effector-silent antibody, antibody fragment, HC constant domain or Fc domain thereof, against human FcRs.

INTRODUCTION

Patients with loss of function mutations in the gene encoding the Nav1.7α channel (SCN9A) show profound insensitivity to pain from birth on. In contrast, gain of function mutations can result in chronic pain disorders. Nav1.7α channels predominantly expressed in peripheral C-fiber nociceptors are therefore a drug target of great interest for treatment of various pain conditions. We have identified ISVDs (Nav1.7 binders) that inhibit Nav1.7α channels with exquisite selectivity over other Nav channel paralogs. Functional inhibitory Nav1.7 activity of the Nav1.7 binders was assessed in automated in vitro patch clamp assays. IC₅₀values in the nanomolar range have been measured. In vivo target modulation in the tissue of interest (peripheral C-fiber nociceptors) was demonstrated in Rhesus microneurography assays. The potential advantages of injectable Nav1.7 binders for the treatment of chronic pain syndromes, such as painful diabetic peripheral neuropathy and osteoarthritis pain, are specificity and extended half-life. Clinical differentiation will be based on improved or comparable efficacy with better side effect profile versus standard of care.

In an embodiment of the invention, any Nav1.7 binder or other binder as set forth herein comprises, where applicable, a substitution of the amino acid at position 11 to the amino acid V and a substitution of the amino acid at position 89 to the amino acid L. In further embodiments, the Nav1.7 binder further includes a substitution of the amino acid at position 110 to the amino acid T, K, or Q. In further embodiments, the amino acid at position 112 is substituted with the amino acid S, K or Q. In each case wherein the numbering is according to the Kabat numbering scheme.

Nav1.7 Sodium Ion Channel

The α-subunits of the Nav1.7 channel are polypeptide chains of 1977 amino acids that are folded into four homologous (but not identical) domains termed DI-DIV that are linked by three intracellular loops (L1-L3). Each domain has six transmembrane segments (S1-S6) with S1-S4 in each domain comprising a voltage sensing domain (VSD), and S5-S6 together with their extracellular linker (including the P-loop) included in the pore domain (PD) (Catterall (2000) Neuron 26:13-25; Guy & Seetharamulu (1986) Proceedings of the National Academy of Sciences of the United States of America 83: 508-512; Noda et al. (1984) Nature 312:121-127). Thus, each α-subunit has four distinct VSDs and four PDs which assemble to form one sodium-selective pore. Sodium is selectivity achieved in the extracellular portion of the pore domain by tight association of the four P-loops that re-enter the membrane between the S5 and S6 segments in DI-DIV and includes several negatively charged residues (aspartic acid and glutamic acid) (Catterall 2000). The human Nav1.7α comprises the amino acid sequence set forth in SEQ ID NO: 1. Domain I of the human Nav1.7α consists of the amino acid sequence shown in SEQ ID NO: 63 and the Domain I S5-S6 loop is shown in SEQ ID NO: 64. The amino acid sequence for the rhesus monkey NAV1.7α is shown in SEQ ID NO: 2, which has 99% identity with the human Nav1.7α. A schematic representation of Nav1.7α is shown in FIG. 32.

Nav1.7 Binders

The present invention provides Nav1.7 binders (e.g., ISVDs) that bind to Nav1.7α and methods of use of the binders for or in the treatment or prevention of disease. In an embodiment of the Nav1.7 binders, the Nav1.7 binders are antagonistic anti-NaV1.7α ISVDs. In further embodiments, the Nav1.7 binder antagonizes the activity of the Nav1.7 channel, for example, by blocking the channel, which may be by physically blocking or closing the Nav1.7 pore to Na⁺ flux or by conformationally changing the Nav1.7 channel to an inactive state.

The Nav1.7 binders include binders that bind to the Domain I S5-S6 loop of the human Nav1.7α comprising amino acids 276 through 331 thereof (e.g., FRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCV (SEQ ID NO: 62)), and heteromeric channels in which the Nav1.7α is complexed with one or more beta subunits such as β1, β2,β3, and/or β4. In an embodiment of the invention, the Nav1.7 binder contacts one or more of the following Nav1.7α amino acid residues: F276, R277, E281, and V331 as shown underlined in the amino acid sequence above. In a further embodiment, the Nav1.7 binder contacts the following four Nav1.7α amino acid residues: F276, R277, E281, and V331. Thus, in particular embodiments, the Nav1.7 binders of the present invention bind to an epitope on Nav1.7α comprising amino acid residues F276, R277, E281, and V331. In a further embodiment, the epitope consists of amino acid residues F276, R277, E281, and V331.

In particular embodiments of the invention, the Nav1.7 binder binds to Nav1.7α having one or more mutations at residue F276, R277, E281, and/or V331 with lower affinity than to human Nav1.7α lacking such mutations. In particular embodiments of the invention, the binder binds to human Nav1.7α comprising one or more mutations at positions Q1530, H1531, and E1534 with a substantially similar affinity to that of human Nav1.7α lacking said mutations. In particular embodiments of the invention, the binder binds to human Nav1.7α comprising mutations at positions Q1530, H1531, and E1534 with a substantially similar affinity to that of human Nav1.7α lacking said mutations. In further embodiments of the invention, the Nav1.7 binder does not bind to rhesus monkey Nav1.7α or binds with a lower affinity than to human Nav1.7α.

In an embodiment of the invention, the Nav1.7 binder binds to human Nav1.7α with substantially similar affinity to human Nav1.7α lacking one more of loops other than the domain 1 S5-S6 loop.

The Nav1.7 binders of the present invention comprise three complementarity determining regions (CDRs) having amino acid sequences selected from the tables below. The CDR amino acid sequences shown in Table 2 and Table 3 are set forth according to the AbM numbering scheme for defining CDR amino acid sequences. A particular CDR amino acid sequence defined by any one of the other schemes advanced for defining CDR amino acid sequences (See Table 1) may have more or less amino acids than shown for CDR amino acid sequences identified according to the AbM numbering scheme but will overlap the CDR amino acid sequences defined according the AbM numbering scheme. Thus, the CDR amino acid sequences shown herein are not to be construed as limiting and any Nav1.7 binder in which the CDR amino acid sequences have been defined by any other numbering scheme will fall within the scope of the Nav1.7 binders of the present invention provided the amino acid sequences for such Nav1.7 binders comprise the amino acid sequences defined for the three CDR amino acid sequences as shown in Table 2 and Table 3. Thus, regardless of the method used to define the CDRs of a Nav1.7 binder (e.g., Kabat, AbM, Clothia, IMGT, Contact, etc.), any Nav1.7 binder that comprises the three amino acid sequences defined for CDR1, CDR2, and CDR3 for any of the Nav1.7 binders shown in Table 2 and Table 3 are Nav1.7 binders of the present invention.

The Nav1.7 binders comprise three CDRs and four Frameworks (FR) in the following alignment FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The Nav1.7 binder CDRs may comprise CDRs comprising the following amino acid sequences.

TABLE 2

Nav1.7 binder
CDR1
CDR2
CDR3

F0103262B06
TRTFSTYAMG
HINFSGSSTRY
ARWVAGPPRYDYEY

(SEQ ID NO: 247)
(SEQ ID NO: 248)
(SEQ ID NO: 249)

F0103262C02
GLPFGLYILG
AISRSGRDTV
DSVPRGTPTITESEYAI

(SEQ ID NO: 250)
(SEQ ID NO: 251)
(SEQ ID NO: 252)

F0103265A11
GMLFNANTQG
FIFSGGYTN
SRY

(SEQ ID NO: 253)
(SEQ ID NO: 254)

F0103265B04
SFIFSNNYME
RITGRGNTN
LWYGGRA

(SEQ ID NO: 256)
(SEQ ID NO: 257)
(SEQ ID NO: 258)

F0103362B08
VRPFSTSAMG
GILWNGIVTY
DRDYGGRSFSAYEYEY

(SEQ ID NO: 259)
(SEQ ID NO: 260)
(SEQ ID NO: 261)

F0103454D07
GGIININYIA
RISSDDTIK
LITPWTGDTRTY

(SEQ ID NO: 262)
(SEQ ID NO: 263)
(SEQ ID NO: 264)

F0103275B05
GSIFNINSMA
SSTNGGSTN
LLQPSIYDISRTY

(SEQ ID NO: 196)
(SEQ ID NO: 198)
(SEQ ID NO: 200)

F0103387G05
GRILRIGYMR
RITDDSATD
LVTASVRGGSIHSGTY

(SEQ ID NO: 201)
(SEQ ID NO: 202)
(SEQ ID NO: 206)

F0103464B09
SRAFIRDVFTG
RIYNGGNTN
SGTINTGREYRSGDY

(SEQ ID NO: 207)
(SEQ ID NO: 213)
(SEQ ID NO: 219)

F0103387G04
GPVFNINKMA
SVTPTGSIS
LLQPDSYSNTRTY

(SEQ ID NO: 221)
(SEQ ID NO: 223)
(SEQ ID NO: 225)

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 247, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 248, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 249.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 250, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 251, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 252.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254, and a CDR3 comprising the amino acid sequence SRY.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 256, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 257, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 258.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 259, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 260, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 261.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 262, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 263, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 264.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 221, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225.

In a further embodiments of the invention, the Nav1.7 binder comprises three CDRs having an amino acid sequence as set forth in Table 3.

TABLE 3

F0103275B05 Family

SEQ

SEQ

SEQ

ID

ID

ID

NO:
CDR1
NO:
CDR2
NO:
CDR3

196
GSIFNINSMA
198
SSTNGGSTN
200
LLQPSIYDISRTY

197
GSIFNINRMA
199

YSTNGGDTN

F0103387G05 Family

SEQ

SEQ

SEQ

ID

ID

ID

NO:
CDR1
NO:
CDR2
NO:
CDR3

201
GRILRIGYMR
202
RITDDSATD
206
LVTASVRGGSIHSGTY

203
RITGGSATG

204
RITDDSATG

205
RITGGSATG

F0103464B09 Family

SEQ

SEQ

SEQ

ID

ID

ID

NO:
CDR1
NO:
CDR2
NO:
CDR3

207
SRAFIRDVFTG
213
RIYNGGNTN
219
SGTINTGREYRSGDY

208
SRAFIRDLFTG
214
RIYNEGNTN

209
SRQFIRDVFTG
215
RIYNEGNTQ

210

HRQFIRDVFTG
216
RIYESGNTQ

211

HRAFIRDVFTG
217
RIYESGNTN

212

HRAFIRDLFTG
218
RIYNEGNTN

F0103387G04 Family

SEQ

SEQ

SEQ

ID

ID

ID

NO:
CDR1
NO:
CDR2
NO:
CDR3

221
GPVFNINKMA
223
SVTPTGSIS
225
LLQPDSYSNTRTY

222
GPVFNINRMA
224

YVTPTGDIS
226
LLQPRRYSNTRTY

227
LLQPDSYSITRTY

228
LLQPRSYSITRTY

229
LLQPRSYSNTRTY

230
LLQPSSYSITRTY

231
LLQPNVYSITRTY

232
LLQPDVYSITRTY

233
LLQPSSYSGTRTY

Amino acid residues in bold face mark those amino acids that are different from the amino acid in the parental sequence.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196 or SEQ ID NO: 197; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 198 or SEQ ID NO: 199; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, or SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, or SEQ ID NO: 212; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, or SEQ ID NO: 218; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201 or SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223 or SEQ ID NO: 224; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 233.

In a further embodiment of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219.

In particular embodiments of the invention, the Nav1.7 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 233.

As recited above, the Nav1.7 binders comprise four frameworks: FR1, FR2, FR3, and FR4 wherein the Nav1.7 binder is a single polypeptide having the structure beginning from the N-terminus FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The numbering of the frameworks may be as shown herein be according to the Kabat numbering scheme and the junction between each framework and a CDR may be defined according to the AbM numbering scheme as shown herein. In particular embodiments, the Nav1.7 binders comprise the VHH2-consensus frameworks FR1, FR2, FR3, and FR4, wherein FR1 has the amino acid sequence set forth in SEQ ID NO: 268, FR2 has the amino acid sequence set forth in SEQ ID NO: 269, FR3 has the amino acid sequence set forth in SEQ ID NO: 270, and FR4 has the amino acid sequence set forth in SEQ ID NO: 271. In further embodiments, each framework may comprise one or more substitutions and or insertions with the proviso that the Nav1.7 binder is capable of binding human Nav1.7α. In further embodiments, frameworks may comprise one or more of the substitutions and/or insertions shown in Table 4 in any combination. In further embodiments, FR1 may comprise one or more of the substitutions shown for FR1 in Table 4. In further embodiments, FR2 may comprise one or more of the substitutions shown for FR2 in Table 4. In further embodiments, FR3 may comprise one or more of the substitutions shown for FR3 in Table 4. In further embodiments, FR4 may comprise one of the substitutions shown for FR4 in Table 4. In a further embodiment, each framework comprises at least one amino acid substitution. In a further embodiment, the Nav1.7 binder comprises at least one substitution and/or insertion shown in Table 4 for each of FR1, FR2, FR3, and FR4. In a further embodiment, the Nav1.7 binder comprises the one substitution or specific substitution and/or insertion combination shown in Table 4 for each of FR1, FR2, FR3, and FR4.

In particular embodiments, the ISVD framework comprises one or more substitutions to minimize binding to pre-existing antibodies. Pre-existing antibodies are antibodies existing in the body of a patient prior to receipt of an ISVD and are immunoglobulins mainly of the IgG class that are present in varying degrees in up to 50% of the human population and that bind to critical residues clustered at the C-terminal region of ISVDs. The ISVDs of the present invention are based, in part, in llama antibodies whose C-terminal constant domains have been removed; thus, exposing the neo-epitopes in the C-terminus of the resulting VHH to pre-existing antibody binding. It has been discovered that the combination of mutations of residues 11 and 89 (e.g., L11V and I89L or V89L) led to a surprising lack of pre-existing antibody binding. Mutations in residue 112 have also been shown to remarkably reduce pre-existing antibody binding. Buyse & Boutton (WO2015/173325) included data showing that the combination of an L11V and V89L mutation provided a remarkable improvement in reducing pre-existing antibody binding compared to an L11V mutation alone or a V89L mutation alone. For example, Table H of Buyse & Boutton on page 97 showed comparative data for an ISVD with a V89L mutation alone (with or without C-terminal extension) and the same ISVD with a V89L mutation in combination with an L11V mutation (again, with or without a C-terminal extension). Also, although generated in two separate experiments, the data shown in Table H for the L11V/V89L combination as compared to the data given in Table B for an L11V mutation alone (in the same ISVD) showed that the pre-existing antibody binding reduction that is obtained by the L11V/V89L combination was greater than that for the L11V mutation alone. Since the llama antibody scaffold structure is known to be very highly conserved, the effect of the mutations at positions 11 and 89 is very likely to exist for any ISVD. Thus, in embodiments herein, the ISVD comprises at least the L11V/V89L substitutions in the framework regions.

In a further embodiment, FR1 comprises at least an L11V substitution and FR3 comprises at least a V89L substitution. In a further still embodiment, the Nav1.7 binder may comprise one of the 125 specific sets of FR1, FR2, FR3, and FR4 combinations shown in Table 4. In any one of the above embodiments, the FR1 may further comprise a Q1E or a Q1D amino acid substitution.

TABLE 4

#
FR1
FR2
FR3
FR4

1
NC
NC
N93R
NC

2
L11V
R39Q
T83R, V89L
NC

3
L11V
NC
R76N, T83R, V89L
NC

4
L11V
NC
T83R, V89L
NC

5
L11V
R39Q
R76N, T83R, V89L
NC

6
L11V
R39Q
R76_V78insT, T83R, V89L
NC

7
L11V
NC
R76_V78insT, T83R, V89L
NC

8
L11V
NC
R76_V78insT, R76N,
NC

T83R, V89L

9
L11V
R39Q
R76_V78insT, R76N,
NC

T83R, V89L

10
L11V
NC
R76N, T83R, V89L, N93R
NC

11
L11V
NC
T83R, V89L, N93R
NC

12
L11V
R39Q
T83R, V89L, N93R
NC

13
L11V
R39Q
R76N, T83R, V89L, N93R
NC

14
D23A
NC
NC
NC

15
D23A
NC
NC
NC

16
L11V, A14P,
G40A, A41P
N82bS, N83R, V89L
R105Q

D23A

17
L11V, A14P,
H37Y, G40A,
N82bS, N83R, V89L
R105Q

D23A
A41P

18
L11V, A14P,
NC
N82bS, N83R, V89L
R105Q

D23A

19
L11V, A14P
H37Y,
N82bS, N83R, V89L
R105Q

20
L11V, A14P
G40A
N82bS, N83R, V89L,
R105Q

L11V, A14P
A41P
N82bS, N83R, V89L
R105Q

21
L11V, A14P
F47L
N82bS, N83R, V89L,
R105Q

22
L11V, A14P
NC
N82bS, N83R, V89L, E93N
R105Q

23
L11V, A14P
NC
N82bS, N83R, V89L
R105Q

24
L11V, A14P,
H37Y, G40A,
N73A, N82bS, N83R,
R105Q

D23A
A41P
V89L

25
L11V, A14P,
H37Y, G40A,
N73Y, N82bS, N83R,
R105Q

D23A
A41P
V89L

26
L11V, A14P,
H37Y, G40A,
N73Q, N82bS, N83R,
R105Q

D23A
A41P
V89L

27
L11V, A14P,
H37Y, G40A,
N82bS, N83R, V89L
R105Q

D23A
A41P

28
L11V, A14P,
H37Y, G40A,
N73Q, N82bS, N83R,
R105Q

D23A
A41P
V89L

29
L11V,
NC
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

30
L11V,
NC
S68T, M77T, T79Y, R81Q,
NC

S82aN, N82bS, K83R,

G88A, V89L

31
L11V,
NC
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L, L93N

32
L11V, T24A
NC
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

33
L11V, T25S
NC
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

34
L11V
R39Q
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

35
L11V
V40A
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

36
L11V
NC
F62S, S68T, T79Y, R81Q,
NC

S82aN, N82bS, K83R,

G88A, V89L

37
L11V
NC
A63V, S68T, T79Y, R81Q,
NC

S82aN, N82bS, K83R,

G88A, V89L

38

NC
L11V, S68T, K76N, T79Y,
NC

R81Q, S82aN, N82bS,

K83R, G88A, V89L

39
L11V
E44Q
S68T, T79Y, R81Q, S82aN,
NC

N82bS, K83R, G88A,

V89L

40
L11V
NC
K83R, V89L
NC

41
L11V
NC
S68T, K83R, V89L
NC

42
L11V
NC
M77T, K83R, V89L
NC

43
L11V
NC
T79Y, K83R, V89L
NC

44
L11V
NC
R81Q, K83R, V89L
NC

45
L11V
NC
S82aN, K83R, V89L
NC

46
L11V
NC
N82bS, K83R, V89L
NC

47
L11V
NC
K83R, G88A, V89L
NC

48
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

49
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

50
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

51
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

52
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

53
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

54
L11V, T24A,
V40A, E44Q,
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

55
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

56
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

57
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

58
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

59
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

60
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

61
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

62
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

63
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

64
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

65
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

66
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

67
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

68
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

69
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

70
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88AV89L

71
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

72
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

73
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

74
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

75
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

76
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

77
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

78
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

79
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

80
L11V, T24A,
V40A, E44Q
F62S, S68T, M77T, T79Y,
NC

T25S

R81Q, S82aN, N82bS,

K83R, G88A, V89L

81
L11V, T24A,
R39Q, V40A,
F62S, S68T, M77T, T79Y,
NC

T25S
E44Q
R81Q, S82aN, N82bS,

K83R, G88A, V89L

82
L11V, T24A,
V40A, E44Q
F62S, A63V, S68T, M77T,
NC

T25S

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

83
L11V, T24A,
R39Q, V40A,
F62S, A63V, S68T, M77T,
NC

T25S
E44Q
T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L

84

N93R

85
L11V, A12V
R39Q
R76_V78insT, T83R,
NC

V89L, N93R

86
L11V, A12V
R39Q
T83R, V89L, N93R
NC

87
L11V, A12V
R39Q
T60A, T83R, V89L, N93R
NC

88
L11V, A12V
R39Q
G73N, T83R, V89L, N93R
NC

89
L11V, A12V
R39Q
R76N, T83R, V89L, N93R
NC

90
L11V, A12V
R39Q
W78V, T83R, V89L, N93R
NC

91
L11V, A12V
R39Q
S79Y, T83R, V89L, N93R
NC

92
L11V, A12V
R39Q
T60A, G73N, R76N,
NC

W78V, S79Y, T83R, V89L,

N93R

93
L11V, A12V
R39Q
T60A, G73N, W78V,
NC

S79Y, T83R, V89L, N93R

94
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

95
L11V, A12V
R39Q
T60A, R76N, W78V, S79Y,
NC

T83R, V89L, N93R

96
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

97
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

98
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R,

99
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

100
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R,

101
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

102
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

103
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

104
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

105
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

106
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

107
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

108
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

109
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R,

110
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

111
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

112
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R,

113
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R

114
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R,

115
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

116
L11V, A12V
R39Q
T60A, G73A, W78V,
NC

S79Y, T83R, V89L, N93R,

117
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

118
L11V, A12V
R39Q
T60A, W78V, S79Y, T83R,
NC

V89L, N93R

119
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

120
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

121
L11V, A12V
R39Q
T60A, G73R, W78V, S79Y,
NC

T83R, V89L, N93R

122
L11V, A12V
R39Q
T60A, D72G, W78V,
NC

S79Y, T83R, V89L, N93R

123
L11V, A12V
R39Q
T60A, D72G, W78V,
NC

S79Y, T83R, V89L, N93R

124
L11V, A12V
R39Q
T60A, D72Q, W78V,
NC

S79Y, T83R, V89L, N93R

125
L11V, A12V
R39Q
T60A, D72Q, W78V,
NC

S79Y, T83R, V89L, N93R

NC—no substitutions and/or insertions; ins—insertion, e.g., R76_V78insT means an insertion between the R at position 76 and the V at position 78; the position numbers are according to the Kabat numbering scheme and the junction between the frameworks and the CDRs are determined according to the AbM numbering scheme.

In a further embodiment of the invention, the Nav1.7 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55.

In a further embodiment of the invention, the Nav1.7 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81.

In a further embodiment of the invention, the Nav1.7 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97.

In a further embodiment of the invention, the Nav1.7 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, and SEQ ID NO: 153.

In a further embodiment of the invention, the Nav1.7 binder comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.

In a further embodiment of the invention, the Nav1.7 binder comprises the amino acid sequence set forth in SEQ ID NO: 96.

In a further embodiment of the invention, the Nav1.7 binder comprises the amino acid sequence set forth in SEQ ID NO: 148.

In a further embodiment of the invention, the Nav1.7 binder comprises the amino acid sequence set forth in SEQ ID NO: 192.

In particular embodiments of the Nav1.7 binders, the N-terminal Glu is substituted with Asp.

Nav1.7 binders of the invention can be fused or linked to one or more other amino acid sequences, chemical entities or moieties by a peptide or non-peptide linker. These other amino acid sequences, chemical entities or moieties can confer one or more desired properties to the resulting Nav1.7 binders of the invention, for example, to provide the resulting Nav1.7 binders of the invention with affinity against another therapeutically relevant target such that the resulting polypeptide becomes “bispecific” with respect to Nav1.7 and that other therapeutically relevant target), or to provide a desired half-life, to provide a cytotoxic effect and/or to serve as a detectable tag or label. Some non-limiting examples of such other amino acid sequences, chemical entities or moieties are:

- one or more suitable peptide or polypeptide linkers (such as a 9GS, 15GS or 35GS linker (any combination of 9, 15, 20 or 35 G and S amino acids such as, for example, GGGGSGGGS (9GS linker; SEQ ID NO: 243), GGGGSGGGGSGGGGSGGGGS (20GS linker; SEQ ID NO: 244) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS linker; SEQ ID NO: 245)), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (50GS linker; SEQ ID NO: 463) or (GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10); and/or
- one or more binding moieties, directed against a target other than Nav1.7 or epitope thereof, for example, against a different epitope of Nav1.7α, Nav1.1α, Nav1.2α, Nav1.3α, Nav1.4α, Nav1.5α, Nav1.6α, Nav1.8α, Nav1.9α, Na_Xalpha subunit, a sodium channel beta subunit (e.g., Navβ1, Navβ2, Navβ3, or Navβ4), a calcium channel or a potassium channel); and/or
- one or more binding domains or binding units that provide for an increase in half-life (for example, a binding domain or binding unit that can bind against a serum protein such as serum albumin, e.g., human serum albumin), e.g., ALB11002; See WO200868280; WO2006122787 or WO2012175400 and/or
- a binding domain, binding unit or other chemical entity that allows for the Nav1.7 binder (e.g., an ISVD such as a Nanobody® ISVD) to be internalized into a desired cell (for example, an internalizing anti-EGFR Nanobody® molecule as described in WO05044858); and/or
- a chemical moiety that improves half-life such as a suitable polyethyleneglycol group (i.e. PEGylation) or an amino acid sequence that provides for increased half-life such as human serum albumin or a suitable fragment thereof (i.e. albumin fusion); and/or
- a payload such as a cytotoxic payload; and/or
- a detectable label or tag, such as a radiolabel or fluorescent label; and/or
- a tag that can help with immobilization, detection and/or purification of the binder (e.g., an ISVD such as a Nanobody® ISVD), such as a HIS_n, wherein n is 6 to 18, or FLAG tag or combination thereof (e.g., SEQ ID NO: 56);
- a tag that can be functionalized, such as a C-terminal GGC tag; and/or
- a C-terminal extension X_(n)(e.g., -Ala), which may be as further described herein for the Nav1.7 binders (e.g., an ISVD such as a Nanobody® ISVD) of the invention and/or as described in WO12175741 or WO2015173325.

Sodium Channel Beta Subunit (Navβ) Binders

The present invention further provides ISVDs that bind the Navβ1 or Navβ2 subunits. These Navβ binders comprise three CDRs having amino acid sequences selected from the table below. The CDR amino acid sequences shown in Table 5 are set forth according to the AbM numbering scheme for defining CDR amino acid sequences. A particular CDR amino acid sequence defined by any one of the other schemes advanced for defining CDR amino acid sequences (See Table 1) may have more or less amino acids than shown for CDR amino acid sequences identified according to the AbM numbering scheme but will overlap the CDR amino acid sequences defined according the AbM numbering scheme. Thus, the CDR amino acid sequences shown herein are not to be construed as limiting and any Navβ binder in which the CDR amino acid sequences have been defined by any other numbering scheme will fall within the scope of the Navβ binders of the present invention provided the amino acid sequences for such Navβ binders comprise the amino acid sequences defined for the three CDR amino acid sequences as shown in Table 5. Thus, regardless of the method used to define the CDRs of a Navβ binder (e.g., Kabat, AbM, Clothia, IMGT, Contact, etc.), any Navβ binder that comprises the three amino acid sequences defined for CDR1, CDR2, and CDR3 for any of the Navβ binders shown in Table 5 are Navβ binders of the present invention.

The Navβ binders comprise three CDRs and four Frameworks (FR) in the following alignment FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The Navβ binder CDRs may comprise CDRs comprising the following amino acid sequences.

TABLE 5

Navß1 binder
CDR1
CDR2
CDR3

F0103478E09
GRAFSTLAMG
ISRNGNNS
ISTPSASHPYVRKESYRY

(SEQ ID NO: 425)
(SEQ ID NO: 426)
(SEQ ID NO: 427)

F0103495F09
GRALSTY AMG
RISRSGITT
DASTNPAGYYLRNRYDY

(SEQ ID NO: 437)
(SEQ ID NO: 438)
(SEQ ID NO: 439)

Navß2 binder
CDR1
CDR2
CDR3

F0103240B04
GGTGRRYAMGW
AIRWSAMTY
TWDYFKYDQVRAYRG

(SEQ ID NO: 422)
(SEQ ID NO: 423)
(SEQ ID NO: 424)

F0103492E09
KSILSFAYMR
SIAIGGATS
PAGQYR

(SEQ ID NO: 428)
(SEQ ID NO: 429)
(SEQ ID NO: 430)

F0103500E09
GRTFSRYQMG
YISWSGSTR
GTAGIISSRPETYDS

(SEQ ID NO: 431)
(SEQ ID NO: 432)
(SEQ ID NO: 433)

F0103505D8
GRTSDLSTMN
RITRRGSTY
ASEMGYHYR

(SEQ ID NO: 434)
(SEQ ID NO: 435)
(SEQ ID NO: 436)

In particular embodiments of the invention, the Navβ1 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 425, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 426, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 427.

In particular embodiments of the invention, the Navβ1 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 437, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 438, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 439.

In particular embodiments of the invention, the Navβ2 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 422, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 423, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 424.

In particular embodiments of the invention, the Navβ2 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 428, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 429, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 430.

In particular embodiments of the invention, the Navβ2 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 431, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 432, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 433.

In particular embodiments of the invention, the Navβ2 binder comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 434, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 435, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 436.

As recited above, the Navβ1 or Navβ2 binders comprise four frameworks: FR1, FR2, FR3, and FR4 wherein the Navβ1 or Navβ2 binder is a single polypeptide having the structure beginning from the N-terminus FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The numbering of the frameworks may be as shown herein be according to the Kabat numbering scheme and the junction between each framework and CDR may be determined by the AbM numbering scheme as shown herein. In particular embodiments, the Nav1.7 binders comprise the VHH2-consensus frameworks FR1, FR2, FR3, and FR4, wherein FR1 has the amino acid sequence set forth in SEQ ID NO: 268, FR2 has the amino acid sequence set forth in SEQ ID NO: 269, FR3 has the amino acid sequence set forth in SEQ ID NO: 270, and FR4 has the amino acid sequence set forth in SEQ ID NO: 271. In further embodiments, each framework may comprise one or more substitutions and or insertions with the proviso that the Navβ1 or Navβ2 binder is capable of binding human Nav1.7α. In further embodiments, frameworks may comprise one or more of the substitutions and/or insertions shown in Table 4 in any combination. In further embodiments, FR1 may comprise one or more of the substitutions shown for FR1 in Table 4. In further embodiments, FR2 may comprise one or more of the substitutions shown for FR2 in Table 4. In further embodiments, FR3 may comprise one or more of the substitutions shown for FR3 in Table 4. In further embodiments, FR4 may comprise one of the substitutions shown for FR4 in Table 4. In a further embodiment, each framework comprises at least one amino acid substitution. In a further embodiment, the Navβ1 or Navβ2 binder comprises at least one substitution and/or insertion shown in Table 4 for each of FR1, FR2, FR3, and FR4. In a further embodiment, the Navβ1 or Navβ2 binder comprises the one substitution or specific substitution and/or insertion combination shown in Table 4 for each of FR1, FR2, FR3, and FR4. In a further embodiment, FR1 comprises at least an L11V substitution and FR3 comprises at least a V89L substitution. In a further still embodiment, the Navβ1 or Navβ2 binder may comprise one of the 125 specific sets of FR1, FR2, FR3, and FR4 combinations shown in Table 4. In any one of the above embodiments, the FR1 may further comprise a Q1E or a Q1D amino acid substitution.

In particular embodiments of the invention, the Navβ1 binder comprises the amino acid sequence set forth in SEQ ID NO: 411.

In particular embodiments of the invention, the Navβ1 binder comprises the amino acid sequence set forth in SEQ ID NO: 415.

In particular embodiments of the invention, the Navβ2 binder comprises the amino acid sequence set forth in SEQ ID NO: 410.

In particular embodiments of the invention, the Navβ2 binder comprises the amino acid sequence set forth in SEQ ID NO: 412.

In particular embodiments of the invention, the Navβ2 binder comprises the amino acid sequence set forth in SEQ ID NO: 413.

In particular embodiments of the invention, the Navβ2 binder comprises the amino acid sequence set forth in SEQ ID NO: 414.

The Navβ binders of the invention can be fused or linked to one or more other amino acid sequences, chemical entities or moieties by a peptide or non-peptide linker. These other amino acid sequences, chemical entities or moieties can confer one or more desired properties to the resulting Navβ binders of the invention, for example, to provide the resulting Navβ binders of the invention with affinity against another therapeutically relevant target such that the resulting polypeptide becomes “bispecific” with respect to Navβ and that other therapeutically relevant target), or to provide a desired half-life, to provide a cytotoxic effect and/or to serve as a detectable tag or label. Some non-limiting examples of such other amino acid sequences, chemical entities or moieties are:

- one or more suitable peptide or polypeptide linkers (such as a 9GS, 15GS or 35GS linker (any combination of 9, 15, 20 or 35 G and S amino acids such as, for example, GGGGSGGGS (9GS linker; SEQ ID NO: 243), GGGGSGGGGSGGGGSGGGGS (20GS linker; SEQ ID NO: 244) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS linker; SEQ ID NO: 245)), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (50GS linker; SEQ ID NO: 463), or (GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10); and/or
- one or more binding moieties, directed against a target other than Navβ or epitope thereof, for example, against a different epitope of Navβ, Nav1.1α, Nav1.2α, Nav1.3α, Nav1.4α, Nav1.5α, Nav1.6α, Nav1.7α, Nav1.8α, Nav1.9α, Na_Xalpha subunit, a sodium channel beta subunit (e.g., Navβ1, Navβ2, Navβ3, or Navβ4), a calcium channel or a potassium channel); and/or
- one or more binding domains or binding units that provide for an increase in half-life (for example, a binding domain or binding unit that can bind against a serum protein such as serum albumin, e.g., human serum albumin), e.g., ALB11002; See WO200868280; WO2006122787 or WO2012175400 and/or
- a binding domain, binding unit or other chemical entity that allows for the Navβ binder (e.g., an ISVD such as a Nanobody® ISVD) to be internalized into a desired cell (for example, an internalizing anti-EGFR Nanobody® molecule as described in WO05044858); and/or a chemical moiety that improves half-life such as a suitable polyethyleneglycol group (i.e. PEGylation) or an amino acid sequence that provides for increased half-life such as human serum albumin or a suitable fragment thereof (i.e. albumin fusion); and/or
- a payload such as a cytotoxic payload; and/or
- a detectable label or tag, such as a radiolabel or fluorescent label; and/or
- a tag that can help with immobilization, detection and/or purification of the binder (e.g., an ISVD such as a Nanobody® ISVD, such as a HIS_n, wherein n is 6 to 18, or FLAG tag or combination thereof (e.g., SEQ ID NO: 56);
- a tag that can be functionalized, such as a C-terminal GGC tag; and/or
- a C-terminal extension X_(n)(e.g., -Ala), which may be as further described herein.

Nav1.7-Navβ Bispecific Binders

The present invention further provides Nav1.7-Navβ bispecific binders comprising at least one Nav1.7 binder and at least one Navβ binder linked together by peptide or polypeptide linker. As used herein, Nav1.7-Navβ bispecific binder refers to binders comprising one or more Nav1.7 binders linked to one or more Navβ binders. In an embodiment, the Nav1.7-Navβ bispecific binders comprise a Nav1.7 ISVD linked via a peptide or polypeptide linker at the C-terminus of the Nav1.7 ISVD to the N-terminus of a Navβ ISVD. In another embodiment, the Nav1.7-Navβ bispecific binders comprise a Navβ ISVD linked via a peptide or polypeptide linker at the C-terminus of the Navβ ISVD to the N-terminus of a Nav1.7 ISVD. The Nav1.7-Navβ bispecific binders are provided as a continuous amino acid sequence.

In particular embodiments, the peptide or polypeptide linker comprises repeating Gly (G) and Ser (S) amino acids to provide for example, 9GS, 15GS, or 35GS peptide or polypeptide linkers (any combination of 9, 15, 20 or 35 G and S amino acids such as, for example, GGGGSGGGS (9GS linker; SEQ ID NO: 243), GGGGSGGGGSGGGGSGGGGS (20GS linker; SEQ ID NO: 244) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS linker; SEQ ID NO: 245)), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (50GS linker; SEQ ID NO: 463), or (GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10).

In particular embodiments, the N-terminal amino acid of the Nav1.7-Navβ bispecific binders is an Asp or Glu amino acid and the C-terminus of the Nav1.7-Navβ bispecific binders comprises a C-terminal extension of one or more Ala amino acids. In particular embodiments, the C-terminal extension consists of one Ala residue.

In particular embodiments of the Nav1.7-Navβ1 bispecific binder, the Navβ binder is a Navβ1 binder or a Navβ2 binder.

In particular embodiments, the Nav1.7-Navβ1 bispecific binder comprises a Navβ1 binder comprising (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 425, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 426, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 427; or (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 437, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 438, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 439.

In a further embodiment, the Nav1.7-Navβ1 bispecific binder comprises a Navβ1 binder comprising the amino acid sequence set forth in SEQ ID NO: 411 or the amino acid sequence set forth in SEQ ID NO: 415.

In particular embodiments, the Nav1.7-Navβ2 bispecific binder comprises a Navβ2 binder comprising (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 422, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 423, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 424; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 428, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 429, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 430; (c) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 431, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 432, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 433; or (d) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 434, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 435, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 436.

In a further embodiment, the Nav1.7-Navβ1 bispecific binder comprises a Navβ2 binder comprising the amino acid sequence set forth in SEQ ID NO: 410, the amino acid sequence set forth in SEQ ID NO: 412, the amino acid sequence set forth in SEQ ID NO: 413, or amino acid sequence set forth in SEQ ID NO: 414.

In particular embodiments, the Nav1.7-Navβ1 bispecific binder comprises a Nav1.7 binder comprising (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196 or SEQ ID NO: 197; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 198 or SEQ ID NO: 199; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, or SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206; (c) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, or SEQ ID NO: 212; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, or SEQ ID NO: 218; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219; (d) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201 or SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223 or SEQ ID NO: 224; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 233; (e) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 206; (0 a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 219; or (g) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 222; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223; and, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 233.

In any one of the foregoing embodiments, the Nav1.7 binder comprising the Nav1.7-Navβ bispecific binder comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55; (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81; (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; (d) an amino acid sequence selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, and SEQ ID NO: 153; (e) an amino acid sequence selected from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195.

In particular embodiments, the Nav1.7 binder comprising the Nav1.7-Navβ bispecific binder comprises the amino acid sequence set forth in SEQ ID NO: 96; the amino acid sequence set forth in SEQ ID NO: 148; or, the amino acid sequence set forth in SEQ ID NO: 192.

In particular embodiments of the Nav1.7 binders or Navβ binders comprising the Nav1.7-Navβ bispecific binder, the N-terminal Glu is substituted with Asp. In particular embodiments, the N-terminal ISVD of the Nav1.7-Navβ binder comprises an Asp amino acid residue at the N-terminus.

Half-Life Extenders (HLE)

The Nav1.7 binders, Navβ binders, and Nav1.7-Navβ bispecific binders of the present invention, may further comprise one or more half-life extenders such as one or more anti-HSA (human serum albumin) binders and/or one or more polyethylene glycol (PEG) molecules.

As discussed herein, the “HSA binders” of the present invention bind to HSA (e.g., an ISVD such as a Nanobody® ISVD) as well as any binder which includes such a molecule that is fused to another binder. An individual HSA binder may be referred to as an HSA binding moiety if it is part of a larger molecule, e.g., a multivalent molecule.

As further described herein, the HSA binders of the invention that are fused to the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder comprise the same combination of CDRs (i.e., CDR1, CDR2 and CDR3) as are present in ALB11002 or comprise the amino acid sequence of ALB11002 (SEQ ID NO: 234).

The present invention also includes Nav1.7 binders, Navβ binders, and Nav1.7-Navβ bispecific binders that further include being linked by a peptide or polypeptide linker to one or more HSA binding moieties which are variants of ALB11002, e.g., wherein the HSA binder comprises CDR1, CDR2 and CDR3 of said ALB11002 variants set forth below in Table 6.

TABLE 6

Human Serum Albumin (HSA) Binders

SEQ

ID

NO:
Description
Sequence

238
ALB11002
EVQLVESGGGXVQPGNSLRLSCAASGFTFSSFGMSW

(may be referred
VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD

to herein as
NAKTTLYLQMNSLRPEDTAXYYCTIGGSLSRSSQGTL

“ALB201”)
VTVSSA;

wherein X at positions 11 and 93 are each L or V.

The CDRs are defined according to the AbM numbering

scheme.

235
HSA-CDR1
GFTFSSFGMS

236
HSA-CDR2
SISGSGSDTL

237
HSA-CDR3
GGSLSR

265
ALB00223
EVOLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSW

VRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRD

NSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTL

VTVSSA

The CDRs are defined according to the AbM numbering

scheme.

267
HSA-CDR1
GFTFRSFGMS

In particular embodiments, the ALB11002 further lacks the C-terminal Alanine (SEQ ID NO: 234). In a further embodiment, the HSA binder comprises the amino acid sequence set forth in SEQ ID NO: 238 but which further comprises an E1D, V11L, and an L93V substitution to provide an HSA binder comprising the amino acid sequence set forth in SEQ ID NO: 240:

EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA.

This embodiment may further lack the C-terminal Alanine to provide the amino acid sequence set forth in SEQ ID NO: 239.

In an embodiment of the invention, the HLE is ALB11 comprising the amino acid sequence:

EVQLVESGGGLVQPGNSLRLSCAASGFTFS SFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA (SEQ ID NO: 242) and in a further embodiment lacks the C-terminal Alanine (SEQ ID NO:241).

In particular embodiments ALB00233 lacks a C-terminal A as shown in SEQ ID NO: 266.

In an embodiment of the invention, the half-life extender is an HSA binder comprising: a CDR1 that comprises the amino acid sequence GFTFSSFGMS (SEQ ID NO: 235) or GFTFRSFGMS (SEQ ID NO: 267); a CDR2 that comprises the amino acid sequence SISGSGSDTL (SEQ ID NO: 236); and a CDR3 that comprises the amino acid sequence GGSLSR (SEQ ID NO: 237).

In an embodiment of the invention, the first amino acid of any of the HSA binders is E and in another embodiment of the invention, the first amino acid of any of the HSA binders is D.

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (50GS linker; SEQ ID NO: 463), or (GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10).

In another embodiment of the invention, the half-life extender is a polyethylene glycol (PEG) moiety appended to the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder to provide a PEGylated Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder. The molecular weight of the polyethylene glycol (PEG) moiety may be about 12,000 daltons or about 20,000 daltons. In an embodiment of the invention, the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder comprises one or more polyethylene glycol molecules covalently attached via a linker (e.g., a C_2-12alkyl such as —CH₂CH₂CH₂—) to a single amino acid residue of a single subunit of the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder, wherein said amino acid residue is the alpha amino group of the N-terminal amino acid residue or the epsilon amino group of a lysine residue. In an embodiment of the invention, the PEGylated binder is: (PEG)_b-L-NH-[binder]; wherein b is 1-9 and L is a C_2-12alkyl linker moiety covalently attached to a nitrogen (N) of the single amino acid residue of the binder. In an embodiment of the invention, the PEGylated binder has the formula: [X-0(CH₂CH₂O) _n]_b-L-NH-[binder], wherein X is H or C_1-4alkyl; n is 20 to 2300; b is 1 to 9; and L is a C_1-11alkyl linker moiety which is covalently attached to the nitrogen (N) of the alpha amino group at the amino terminus of one binder subunit; provided that when b is greater than 1, the total of n does not exceed 2300. See, for example, U.S. Pat. No. 7,052,686, which is incorporated herein by reference in its entirety.

To PEGylate a Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder, typically the binder is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the binder. In particular embodiments, the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the binder to be PEGylated is an aglycosylated binder. Methods for PEGylating proteins are known in the art and can be applied to the binder of the invention. See, e.g., EP0154316 and EP0401384, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder is fused at the C-terminus to an HC constant domain of Fc domain thereof domain. In a particular embodiment, the HC domain or Fc domain thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype. The amino acid sequences of the IgG1, IgG2, and IgG4 isotype HC constant domains are set forth in SEQ ID NO: 469, SEQ ID NO: 476, and SEQ ID No: 482, respectively. In the embodiments herein, the Fc domain may comprise the CH2 and CH3 domains of the HC constant domain. In particular embodiments, the Fc domain may further comprise the hinge region between the CH1 and CH2 domains or the hinge region comprising one or amino acid deletions. In exemplary embodiments, Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are fused to an HC domain or Fc domain thereof of the IgG1, IgG2, or IgG4 isotype. In particular embodiments, the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are fused to the N-terminus of an HC domain or Fc domain thereof. In particular embodiments, the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are fused to the C-terminus of an HC domain or Fc domain thereof.

Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders of the present invention further include ISVDs that are fused or linked to an effector-silent HC constant domain or Fc domain thereof The effector-silent HC constant domain or Fc domain has been modified such that it displays no measurable binding to one or more FcRs or displays reduced binding to one or more FcRs compared to that of an unmodified HC constant domain or Fc domain of the same IgG isotype. The effector-silent HC constant domain or Fc domain may in further embodiments display no measurable binding to each of FcγRIIIa, FcγRIIa, and FcγRI or display reduced binding to each of FcγRIIIa, FcγRIIa, and FcγRI compared to that of an unmodified antibody of the same IgG isotype. In particular embodiments, the effector-silent HC constant domain or Fc domain is a modified human HC constant domain or Fc domain.

In particular embodiments, the effector-silent HC constant domain or Fc domain thereof comprises an Fc domain of an IgG1 or IgG2, IgG3, or IgG4 isotype that has been modified to lack N-glycosylation of the asparagine (Asn) residue at position 297 (Eu numbering system) of the HC constant domain. The consensus sequence for N-glycosylation is Asn-Xaa-Ser/Thr (wherein Xaa at position 298 is any amino acid except Pro); in all four isotypes the N-glycosylation consensus sequence is Asn-Ser-Thr. The modification may be achieved by replacing the codon encoding the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, for example Ala, Asp, Gln, Gly, or Glu, e.g. N297A, N297Q, N297G, N297E, or N297D. Alternatively, the codon for Ser at position 298 may be replaced with the codon for Pro or the codon for Thr at position 299 may be replaced with any codon except the codon for Ser. In a further alternative each of the amino acids comprising the N-glycosylation consensus sequence is replaced with another amino acid. Such modified IgG molecules have no measurable effector function. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations. In further embodiments, such IgGs modified to lack N-glycosylation at position 297 may further include one or more additional mutations disclosed herein for eliminating measurable effector function.

An exemplary IgG1 HC constant domain or Fc domain thereof mutated at position 297, which abolishes the N-glycosylation of the HC constant domain, is set forth in SEQ ID NO: 474, an exemplary IgG2 HC constant domain mutated at position 297, which abolishes the N-glycosylation of the HC constant, is set forth in SEQ ID NO: 480, and an exemplary IgG4 HC constant domain mutated at position 297 to abolish N-glycosylation of the HC constant domain is set forth in SEQ ID NO: 485. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the HC constant domain or Fc domain thereof of the IgG1 IgG2, IgG3, or IgG4 HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu numbering) and wherein said HC constant domain is effector-silent. In particular embodiments, the modified IgG1 further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the HC constant domain or Fc domain thereof comprises L234A, L235A, and D265S substitutions (wherein the positions are identified according to Eu numbering). In particular embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu numbering). These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (2000), each of which is incorporated herein by reference in its entirety).

In particular embodiments of the above, the HC constant domain or Fc domain thereof comprises an L234A/L235A/D265A; L234A/L235A/P329G; L235E; D265A; D265A/N297G; or V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions, wherein the positions are identified according to Eu numbering. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the effector-silent HC constant domain or Fc domain thereof comprises an IgG1 isotype, in which the Fc domain of the HC constant domain has been modified to be effector-silent by substituting the amino acids from position 233 to position 236 of the IgG1 with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgG4 HC, wherein the positions are identified according to Eu numbering (Armour et al., Eur. J. Immunol. 29(8):2613-24 (1999); Shields et al., J. Biol. Chem. 276(9):6591-604(2001)). In particular embodiments, the modified IgG1 further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the effector-silent HC constant domain or Fc domain thereof is a hybrid human immunoglobulin HC constant domain, which includes a hinge region, a CH2 domain and a CH3 domain in an N-terminal to C-terminal direction, wherein the hinge region comprises an at least partial amino acid sequence of a human IgD hinge region or a human IgG1 hinge region; and the CH2 domain is of a human IgG4 CH2 domain, a portion of which, at its N-terminal region, is replaced by 4-37 amino acid residues of an N-terminal region of a human IgG2 CH2 or human IgD CH2 domain. Such hybrid human HC constant domain is disclosed in U.S. Pat. No. 7,867,491, which is incorporated herein by reference in its entirety.

In particular embodiments, the effector-silent HC constant domain or Fc domain thereof is an IgG4 HC constant domain in which the serine at position 228 according to the Eu system is substituted with proline, see for example SEQ ID NO: 52. This modification prevents formation of a potential inter-chain disulfide bond between the cysteines at positions Cys226 and Cys229 in the EU numbering scheme and which may interfere with proper intra-chain disulfide bond formation. See Angal et al. Mol. Imunol. 30:105 (1993); see also (Schuurman et al., Mol. Immunol. 38: 1-8, (2001)). In further embodiments, the IgG4 constant domain includes in addition to the S228P substitution, a P239G, D265A, or D265A/N297G amino acid substitution, wherein the positions are identified according to Eu numbering. In particular embodiments of the above, the IgG4 HC constant domain is a human HC constant domain. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

Exemplary IgG1 HC constant domains comprise an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, and SEQ ID NO: 475.

Exemplary IgG2 HC constant domains comprise an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, and SEQ ID NO: 480.

Exemplary IgG4 HC constant domains comprise an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO: 483, SEQ ID NO: 484, and SEQ ID NO: 485.

The particular embodiments, the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder is linked to the HC constant domain or Fc domain thereof by a peptide or polypeptide linker to provide a fusion protein comprising the structure binder-linker-HC constant domain or Fc domain thereof or HC constant domain-linker-binder wherein binder refers to Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder. The Fc domain thereof as used herein includes embodiments lacking the hinge region and embodiments wherein the Fc comprises one or amino acids of the hinge region.

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (50GS linker; SEQ ID NO: 463), or (GGGGS (SEQ ID NO: 246)) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

In particular embodiments, the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are fused to the N-terminus of an effector-silent HC domain or Fc domain thereof. In particular embodiments, the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are fused to the C-terminus of an effector-silent HC domain or Fc domain thereof.

In particular embodiments, the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders are linked to the N-terminus of an effector-silent HC domain or Fc domain thereof by a non-peptide linker, which in particular embodiments, may be a non-peptide polymer. The non-peptide polymer refers to a biocompatible polymer to which at least two repeat units are conjugated, and the repeat units are interconnected by random covalent bonds other than peptide bonds. The non-peptide polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer between ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymer, chitins, hyaluronic acid, and a combination thereof, and preferably, polyethylene glycol. The derivatives known in the art and the derivatives that can easily be prepared using the technology in the art are also included in the scope of the present invention. In particular embodiments, the non-peptide linker comprises polyethylene glycol, which in particular embodiments may be 3,400 daltons. Conjugates comprising a heterologous protein conjugated to an Fc domain by a non-peptide linker have been disclosed in U.S. Pat. Nos. 7,636,420; 7,737,260; 7,968,316; 8,029,789; 8,110,665; 8,124,094; 8,822,650; 8,846,874; 9,394, 546; 10,071,171; 10,272,159; and 10,973,881, each of which is incorporated herein by reference in its entirety.

In particular embodiments, the HC constant domain or Fc domain conjugates form a homodimer wherein each HC constant domain or Fc domain conjugates comprising the homodimer is fused or conjugated to the same binder selected from Nav1.7 binder, Navβ binder, and Nav1.7-Navβ bispecific binder. In particular embodiments, the HC constant domain or Fc domain conjugates form a heterodimer wherein a HC constant domain or Fc domain conjugate comprising the heterodimer is fused or conjugated to a binder selected from Nav1.7 binder, Navβ binder, and Nav1.7-Navβ bispecific binder and a second HC constant domain or Fc domain conjugate comprising the heterodimer is fused or conjugated to a binder selected from Nav1.7 binder, Navβ binder, and Nav1.7-Navβ bispecific binder that is not fused or conjugated to the first HC constant domain or Fc domain conjugate. In particular embodiments, the HC constant domain or Fc domain conjugate form a heterodimer wherein a first HC constant domain or Fc domain conjugate comprising the heterodimer is fused or conjugated to a binder selected from Nav1.7 binder, Navβ binder, and Nav1.7-Navβ bispecific binder and the second HC constant domain or Fc domain is not fused or conjugated to a Nav1.7 binder, Navβ binder, and Nav1.7-Navβ bispecific binder. In particular embodiments, the second HC constant domain or Fc domain is fused or conjugated to a heterologous protein, which may be the Fab of an antibody or ISVD other than a Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder; a heterologous protein, polypeptide, or peptide; or a small molecule. HC constant domain and Fc domain heterodimers have been disclosed in WO9627011; WO9850431; WO9929732; WO2009089004; WO2013055809; WO2013063702; WO2014145907; and WO2014084607, each of which is incorporated herein by reference in its entirety.

In particular embodiments of the invention, the HC constant or Fc domains as disclosed herein may comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

C-Terminal Extensions

The present invention further provides Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders that comprise a C-terminal extension. The present invention provides, for example, C-terminal extensions such as X(n), wherein X and n can be as follows:

- (a) n=1 and X=Ala;
- (b) n=2 and each X=Ala;
- (c) n=3 and each X=Ala;
- (d) n=2 and at least one X=Ala (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (e) n=3 and at least one X=Ala (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (f) n=3 and at least two X=Ala (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (g) n=1 and X=Gly;
- (h) n=2 and each X=Gly;
- (i) n=3 and each X=Gly;
- (j) n=2 and at least one X=Gly (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (k) n=3 and at least one X=Gly (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (l) n=3 and at least two X=Gly (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
- (m) n=2 and each X=Ala or Gly;
- (n) n=3 and each X=Ala or Gly;
- (o) n=3 and at least one X=Ala or Gly (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile); or
- (p) n=3 and at least two X=Ala or Gly (with the remaining amino acid residue(s) X being independently chosen from any naturally occurring amino acid but preferably being independently chosen from Val, Leu and/or Ile);
  
  with aspects (a), (b), (c), (g), (h), (i), (m) and (n) being preferred, with aspects in which n=1 or 2 being preferred and aspects in which n=1 being preferred.

Some specific, but non-limiting examples of useful C-terminal extensions are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA or GAG.

In an embodiment of the invention, any C-terminal extension present in a Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder does not contain a free cysteine residue (unless said cysteine residue is used or intended for further functionalization, for example for PEGylation).

Conjugates

The Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders disclosed herein may also be conjugated to a chemical moiety. Such conjugated binders are an embodiment of the present invention. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor. In particular embodiments, the chemical moiety is a polymer that increases the half-life of the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder in the body of a subject. Suitable polymers include, but are not limited to, hydrophilic polymers, which include but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders disclosed herein may also be conjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The Nav1.7 binders may also be conjugated with fluorescent or chemiluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

The Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.

Any method known in the art for conjugating a Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binder to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating binders are conventional and very well known in the art.

The present invention further provides nucleic acid molecules encoding any one of the Nav1.7 binders, Navβ binders, or Nav1.7-Navβ bispecific binders disclosed herein. In particular embodiments, the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NO: 273-283. In particular embodiments, the nucleic acid molecule encoding the Nav1.7 binder comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NO: 284-421. In particular embodiments, the nucleic acid molecule encoding the Navβ binder comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NO: 456-461.

The following examples are intended to promote a further understanding of the present invention. The amino acid sequences for the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binders and nucleic acid sequences encoding the Nav1.7 binder, Navβ binder, or Nav1.7-Navβ bispecific binders that are disclosed in the following examples are provided in Table 56. Various embodiments of the aforementioned binders comprise an amino acid sequence set forth in Table 56.

Example 1

Generation of Stable Recombinant huNav1.7α Cell Lines

Different stable CHO FlpIn (ThermoFisher Scientific, catalog #R758-07) or HEK FlpIn (ThermoFisher Scientific, catalog #R750-07) transgenic cell lines were generated according to the manufacturer's instructions. To this purpose, different Nav1.7α constructs (human or rhesus) were cloned into pcDNA5/FRT (ThermoFisher Scientific, catalog #V601020). The amino acid sequences for huNav1.7α, rhNav1.7α, huNav1.1α, huNav1.2α, huNav 1.3α, huNav1.4α, huNav1.5α, huNav1.6α, and huNav1.8α are set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. The generation of HEK293 cell lines stably expressing huNav1.7a with and without the human β subunit is detailed elsewhere (Schmalhofer et al. Mol Pharmacol 74:1476-1484, 2008). HEK cell lines expressing huNav1.1α, huNav1.2α, huNav1.3α, huNav1.4α, huNav1.5α, huNav1.6α, or huNav1.8α were constructed.

A detailed sequence comparison of the different extra-cellular loops (ECLs) of huNav1.7a to their ortholog and paralog counterparts is shown in FIGS. 2A-2B. Different splice variants of Nav1.7α exist that through interaction with β1 impact on the electrophysiological properties of the channel (Chatelier et al. 2008 J Neurophysiol 99: 2241; Farmer et al. 2012 PLoS ONE 7: e41750). The 5N11S variant of huNav1.7a (FIG. 32) was used consistently throughout the examples. The major technical drawbacks of Nav1.7α as a target for biologicals are its poor cell surface expression level combined with a limited accessibility to the extracellular surface.

For various experiments set forth in the examples, the Nav constructs where indicated were fused at the C-terminus via a P2A viral peptide linker (SEQ ID NO: 43) to a single polypeptide encoding sodium channel beta subunits β1 (SEQ ID NO: 40), β2 (SEQ ID NO: 41), and β3 (SEQ ID NO: 42) in tandem in which each β subunit is separated from the preceding β subunit by a P2A viral peptide linker (referred to herein as β1-β2-β3; See SEQ ID NO:21). The P2A peptide linker facilitates a co-translational cleavage event that effectively liberates polypeptides N-terminal and C-terminal to it.

Plasmid Constructs and Expression Vectors

Table 7 gives an overview of all plasmid constructs and expression vectors.

TABLE 7

Overview plasmid DNA constructs

Plasmid ID
Description

pCMV6-AC-Myc-DDK-
Origene clone with wild type huNav1.7α sequence

NM_002977.1
(NM_002977.1)

pFRT/lacZEO
Basic vector for generation of Flp-In compatible cell

backgrounds

pcDNA3.1-CO_huNav1.7
Codon-optimized human source sequence

pcDNA3.1-CO_rhNav1.7
Codon-optimized rhesus source sequence

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim14
sequence (ECLs and transmembrane helices from Nav1.7α

ICLs from Nav1.5α) | combines native extracellular

conformation of Nav1.7α with increased expression levels of

Nav1.5α

pJTI-R4-DEST-
Codon-optimized huNav1.7α/Nav1.5α chimera and the

CO_huNav157chim14-
Navβ1-β3 subunits (β1-β2-β3) as picoRNA viral fusion source

PV_SCN1B-SCN2B-SCN3B
sequence

pJTI-R4-DEST-
Codon-optimized huNav1.7α and the Navβ1-β3 subunits (β1-

CO_huNav1.7-PV_SCN1B-
β2-β3) as picoRNA viral fusion source sequence

SCN2B-SCN3B

pVAX1-NM_002977.1
Vector for DNA immunizations with wild type huNav1.7α

sequence

pcDNA3.1/Hygro-
Vector for cell line transfections with wild type huNav1.7α

NM_002977.1
sequence

pcDNA5/FRT-NM_002977.1
Vector for Flp-In cell line transfections with wild type

huNav1.7α sequence

pcDNA5/FRT-CO_huNav1.7
Vector for Flp-In cell line transfections with codon-optimized

huNav1.7α sequence

pVAX1-CO_huNav1.7
Vector for DNA immunizations with codon-optimized

huNav1.7α sequence

pcDNA5/FRT-
Vector for Flp-In cell line transfections with huNav157

huNav157chim14
chimera 14

pVAX1-
Vector for DNA immunizations with huNav157 chimera 14

CO_huNav157chim14

pcDNA5/FRT-
Vector for Flp-In cell line transfections with huNav157

CO_huNav157chim14-
chimera 14 and the Navβ1-β3 subunits (β1-β2-β3)

PV_SCN1B-SCN2B-SCN3B

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7-PV_SCN1B-
huNav1.7α sequence and the Navβ1-β3 subunits (β1-β2-β3)

SCN2B-SCN3B

pcDNA5/FRT-CO_huNav1.7
Vector for Flp-In cell line transfections with codon-optimized

(P149_D150insFLAG)
huNav1.7α with triple FLAG tag inserted between aa 149 and

150 (S1 of Domain 1) for cell surface expression detection via

tag

pcDNA5/FRT-CO_huNav1.7
same as above but triple FLAG inserted between aa 148 and

(P148_P149insFLAG)
149

pcDNA3.1-CO_huNav1.5α
Codon-optimized human Nav1.5α source sequence

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.5-PV_SCN1B-
huNav1.5α sequence and the Navβ1-β3 subunits (β1-β2-β3) to

SCN2B-SCN3B
be used as controls in selections and screening

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim1*
sequence (DI, DII and DIII from Nav1.7α, DIV from

Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7/Nav1.5α chimera source

CO_huNav157chim2*
sequence (DI, DII and DIV from Nav1.7α, DIII from

Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim3*
sequence (DI, DIII and DIV from Nav1.7α, DII from

Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim4*
sequence (DII, DIII and DIV from Nav1.7α, DI from

Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim5*
sequence (DI, DII and DIII from Nav1.5α, DIV from

Nav1.7α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim6*
sequence (DI, DII and DIV from Nav1.5α, DIII from

Nav1.7α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim7*
sequence (DI, DIII and DIV from Nav1.5α, DII from

Nav1.7α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim8*
sequence (DII, DIII and DIV from Nav1.5α, DI from

Nav1.7α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim9*
sequence (DI, DII, DIII and DIV VSD from Nav1.7α, DIV

S5-S6 from Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim10*
sequence (DI, DII, DIII VSD and DIV from Nav1.7α, DIII

S5-S6 from Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim11*
sequence (DI, DI VSD, DIII and DIV from Nav1.7α, DII S5-

S6 from Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim12*
sequence (DI VSD, DII, DIII and DIV from Nav1.7α, DI S5-

S6 from Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim18*
sequence (DI, DII, DIII and DIV S5-S6 from Nav1.7α, DIV

VSD from Nav1.5α)

pcDNA3.1-
Codon-optimized huNav1.7α/Nav1.5α chimera source

CO_huNav157chim22*
sequence (DI, DII, DIII and DIV S3-S6 from Nav1.7α, DIV

S1-S2 from Nav1.5α)

pcDNA5/FRT-CO_rhNav1.7-
Vector for Flp-In cell line transfections with codon-optimized

PV_SCN1B-SCN2B-SCN3B
rhNav1.7α sequence and the human Navβ1-β3 subunits (β1-

β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(N146S, V194I,
huNav1.7α sequence containing all DI polymorphisms of

F276V, R277Q, E281V, V331M,
rhNav1.7α and the human Navβ1-β3 subunits (β1-β2-β3)

E504D, D507E, S508N, N533S)-

PV_SCN1B-SCN2B-SCN3B

pcDNA5/FRT-CO_rhNav1.7-
Vector for Flp-In cell line transfections with codon-optimized

PV_rhSCN1B-rhSCN2B-
rhNav1.7α sequence and the rhesus Navβ1-β3 subunits (β1-

rhSCN3B
β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(F276V)-
huNav1.7α sequence containing extracellular DI rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism F276V and the human Navβ1-β3 subunits (β1-

β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(R277Q)-
huNav1.7α sequence containing extracellular DI rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism R277Q and the human Navβ1-β3 subunits (β1-

β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(E281V)-
huNav1.7α sequence containing extracellular DI rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism E281V and the human Navβ1-β3 subunits (β1-

β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(V331M)-
huNav1.7α sequence containing extracellular DI rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism V331M and the human Navβ1-β3 subunits

(β1-β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(Q1530P)-
huNav1.7α sequence containing extracellular DIV rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism Q1530P and the human Navβ1-β3 subunits

(β1-β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(H1531Y)-
huNav1.7α sequence containing extracellular DIV rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism H1531Y and the human Navβ1-β3 subunits

(β1-β2-β3)

pcDNA5/FRT-
Vector for Flp-In cell line transfections with codon-optimized

CO_huNav1.7(E1534D)-
huNav1.7α sequence containing extracellular DIV rhNav1.7α

PV_SCN1B-SCN2B-SCN3B
polymorphism E1534D and the human Navβ1-β3 subunits

(β1-β2-β3)

*huNav157 chimeras are schematically drawn in FIG. 16.

Generation of HEK293T Cells, Transiently Transfected with Different huNav1.7α Constructs

To this purpose, different Nav1.7α constructs were cloned into pcDNA3.1 (ThermoFisher Scientific, catalog #V79020) and plasmid DNA was prepared from Escherichia coli TOP10 cells. HEK293T cells were seeded at a concentration of 1.5×10⁶per T75 flask and incubated overnight at 37° C. in DMEM (Dulbecco's modified Eagle's medium; Gibco, catalog #31966) supplemented with 10% FBS (fetal bovine serum, Sigma. Catalog #F7524). The medium was then replaced by Opti-MEM medium (Gibco, catalog #31985). A mixture of 9 μg plasmid DNA, 27 μL, Fugene 6 (Promega, catalog #E2691) in a final volume of 1 mL Opti-MEM was incubated for 15 min at room temperature and then added to the cells. After 3 hours incubation at 37° C., 10 mL of DMEM supplemented with 20% FBS was added and incubation continued. After 48 hours, cells were washed with phosphate buffered saline (PBS) and resuspended with 4 mL of trypsin EDTA (Gibco, catalog #25200-056) followed by addition of 6 mL DMEM medium supplemented with 10% FBS.

Membrane Preparations

On Day 1, suspend pellet in 3 mL HB (250 mM Sucrose, 25 mM HEPES, pH 7.5)+μL Mammalian Protease Inhibitor cocktail+30 μL Benzonase/Nuclease-Dnase (25 U/μL) PER 1 billion cells; dounce homogenize with 5 strokes of a Type B/tight fit pestle (glass homogenizer); transfer homogenized cells to Nalgene 3119-0050 Oak Ridge centrifuge tubes and centrifuge at 5 k×g (6,025 rpm) for 30 minutes at 4° C. Collect supernatant fraction (and store on ice (pellet P1). Suspend pellet in 2 mL HB. Repeat dounce homogenization and transfer homogenized cells to fresh 50 mL falcon tubes. Increase the volume to 50 mL with HB. Centrifuge at 2 k xg (3,161 rpm) in for 15 minutes at 4° C.; collect the supernatant fraction, and pool with supernatant fraction collected above (P1). Suspend pellet in 2 mL HB. Repeat dounce homogenization. Increase volume to 50 mL with HB. Repeat 2K xg centrifugation. Collect the supernatant fraction and pool with the supernatant fraction collected above (P1). Transfer pooled supernatant fraction to fresh Nalgene tubes. Fill to fill line with HB. (P1) Suspend remaining pellet and transfer to fresh Nalgene tube. Fill to fill-line with HB to produce pellet 2 (P2). Centrifuge P1 & P2 at 39,800 xg (17 k rpm) for 45 minutes at 4° C. Keep 1 mL of supernatants (s1a+s2a). Store in −80° C. and decant remainder of supernatant fractions. Suspend pellets (P1+P2) in 0.1 M FB (100 mM NaCl, 25 mM Tris-HCl pH7.5). Repeat centrifugation at 39.8 k xg for 45 minutes at 4° C. Keep 1 mL of supernatants (s1b+s2b). Store at −80° C. Decant remainder of supernatants. Store pellets (P1+P2) on ice in 4° C. overnight.

On Day 2, suspend pellets in 1.5 M FB (1.5 M NaCl, 25 mM Tris-HCl pH7.5); dounce homogenize with 5 strokes of a Type B/tight fit pestle (glass homogenizer); transfer pellet to Nalgene 3119-0050 tube(s) and fill to fill line with 1.5 M FB; centrifuge at 39.8 k xg for 45 min at 4C; remove supernatant fraction and store pellets at −80° C. (SA).

Pool like pellets in 5-10 mL 1.5 M FB; dounce homogenize with 5 strokes of a Type B/tight fit pestle (glass homogenizer); return membrane to Nalgene tube and again fill to fill line with 1.5 M FB; repeat centrifuge at 39,800 xg (17 k rpm) for 45 minutes at 4° C. Remove supernatant fraction and store pellets at −80° C. (SB).

Suspend pellets in 5-10 mL 0.1 M FB; repeat dounce homogenization; return membrane to Nalgene tube and fill to fill line with 0.1 M FB; Centrifuge a 3rd time at 39,800 xg (17 k rpm) for 45 minutes at 4° C. Remove supernatant fraction and store pellet at −80° C. (SC).

Suspend pellets in 0.1 M FB; dounce homogenize with 5 strokes of a Type B/tight fit pestle (glass homogenizer); determine protein concentrations via Bradford assay; if desired, adjust concentration with 0.1 M FB; aliquot mem preparations, freeze on dry ice and store at 80° C.

Binding FACS

Binding of the ISVDs to cell-expressed Nav1.7α was detected via murine anti-Flag (Sigma, catalog #F1804). Briefly, cells were resuspended in FACS buffer (PBS, 10% FBS, NaN₃) and transferred to a 96-well V-bottom plate at 1×10 5 cells/well. Purified FLAG3-tagged ISVD was diluted in FACS buffer and added to the cells for 30 minutes at 4° C. ISVD binding was detected by resuspending the samples subsequently in 100 μL murine anti-Flag at 1 μg/mL and 100 μL APC-labelled goat anti-mIgG (Jackson ImmunoResearch, catalog #115-135-164). Prior to the read-out, the samples were resuspended in 1 μg/mL propidium iodide (Sigma, catalog #P4170) to exclude dead cells. Between each step, the cells were centrifuged for 5 minutes at 200 grams and washed with 100 μL/well FACS buffer. An alternative approach used PE-labelled goat anti-murine IgG (Jackson ImmunoResearch, catalog #115-116-071) as detection antibody and 5 nM TOPRO3 (Molecular probes, catalog #T3605) as dead dye.

Control antibodies were detected as follows. Murine anti-Nav1.7α mAb S68-6 (Abcam, catalog #ab85015) was detected by PE-conjugated goat anti-murine IgG (Jackson ImmunoResearch, catalog #115-116-071) after fixation and permabilization of the cells with FIX & PERM kit according to the manufacturer's instructions (ThermoFisher Scientific, catalog #GAS003). Rabbit anti-Nav1.5α pAb (Alomone Labs, catalog #ASC-013) was detected with PE-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, catalog #711-116-152) after fixation and permabilization of the cells with FIX & PERM kit according to the manufacturer's instructions (ThermoFisher Scientific, catalog #GAS003). Rabbit anti-human 4 pAb (ThermoFisher Scientific, catalog #PAS-24142) was detected with PE-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, catalog #711-116-152).

Immunizations

After approval of the Ethical Committee of the faculty of Veterinary Medicine (University Ghent, Belgium) or the Ethical Committee of the Ablynx Camelid Facility (LA1400575), 3 camelids were immunized with a CMV-promoter based DNA vector encoding codon optimized huNav1.7α, followed by codon optimized huNav157 chimera 14 DNA and membrane extracts prepared from recombinant HEK293 cells expressing huNav1.7α together with Navβ1, Navβ2 and Navβ3 (as described above).

Cloning of Heavy Chain-Only Antibody Fragment Repertoires and Preparation of Phage

Following the final immunogen injection, blood samples were collected. From these blood samples, peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, US). From the PBMCs, total RNA was extracted and used as starting material for RT-PCR to amplify the VHH/ISVD-encoding DNA segments, essentially as described in WO05044858. Subsequently, phages were prepared according to standard protocols (see for example the prior art and applications filed by Ablynx N.V. cited herein) and stored after filter sterilization at 4° C. for further use.

Selection of Nav1.7α Specific ISVDs Via Phage Display

VHH repertoires obtained from all camelids and cloned as phage library were subjected for two or three consecutive selection rounds to proteoliposome (PL) (5 μg/mL) or amphipol (amphipathic surfactant for maintaining solubilized membrane proteins in detergent-free solutions, catalog #A835, Anatrace) preparations (5 μg/mL) derived from HEK293 cells recombinantly expressing huNav1.7α together with Navβ1, Navβ2, and Navβ3 subunits (β1-β2-β3). Each selection round was performed in the presence of the following competing agents: 100 μg/mL of in house produced membrane extracts from HEK293 cells and 100 nM each of recombinant Navβ1 (Abnova, catalog #H00006324-P01), Navβ2 (Sino Biological, catalog #13859-H02H) and Navβ3 (Sino Biological, catalog #13500-H02H). After antigen incubation of the libraries and extensive washing; bound phage were eluted with trypsin (1 mg/mL) for 15 minutes and then the protease activity was immediately neutralized by applying 0.8 mM protease inhibitor ABSF. As a control, selections with in-house produced membrane extracts from HEK293 cells or without antigen were performed in parallel. Phage outputs were used to infect E. coli TG1 for analysis of individual VHH clones. Periplasmic extracts were prepared according to standard protocols (see for example WO03035694, WO04041865, WO04041863, WO04062551).

Generation of ISVD Expression Constructs

Sequence analysis of ISVDs from phage display selection outputs was done according to commonly known procedures (Pardon et al., Nat Protoc 9: 674 (2014)). ISVD-containing DNA fragments, obtained by PCR with specific combinations of forward FR1 and reverse FR4 primers each carrying a unique restriction site, were digested with the appropriate restriction enzymes and ligated into the matching cloning cassettes of ISVD expression vectors (described below). The ligation mixtures were then transformed to electrocompetent Escherichia coli TG1 (60502, Lucigen, Middleton, WI) cells which were then grown under the appropriate antibiotic selection pressure. Resistant clones were verified by Sanger sequencing of plasmid DNA (LGC Genomics, Berlin, Germany). Monovalent ISVDs were expressed in E. coli TG1 from a plasmid expression vector containing the lac promoter, a resistance gene for kanamycin, an E. coli replication origin and an ISVD cloning site preceded by the coding sequence for the OmpA signal peptide. In frame with the ISVD coding sequence, the vector codes for a C-terminal FLAG3 (or CMYC3) and HIS6 tag. The signal peptide directs the expressed ISVDs to the periplasmic compartment of the bacterial host.

Unless specified otherwise, the tested clones herein comprise the ISVD amino acid sequence shown for it in Table 56 further fused at the C-terminus to a FLAG-HIS6 polypeptide (SEQ ID NO: 56) or HIS6. The amino acid positions in the ISVDs disclosed herein are numbered according to the Kabat numbering scheme.

Generic Expression and Purification of ISVDs

E. coli TG-1 cells containing the ISVD constructs of interest were grown for 2 hours at 37° C. followed by 29 hours at 30° C. in baffled shaker flasks containing “5052” auto-induction medium (0.5% glycerol, 0.05% glucose, 0.2% lactose+3 mM MgSO₄). Overnight frozen cell pellets from E. coli expression cultures are then dissolved in PBS (1/12.5^thof the original culture volume) and incubated at 4° C. for one hour while gently rotating. Finally, the cells were pelleted down once more, and the supernatant containing the proteins secreted into the periplasmic space was stored for further purification. HIS6-tagged ISVDs were purified by immobilized metal affinity chromatography (IMAC) on either Ni-Excel (GE Healthcare) or Ni-IDA/NTA (Genscript) resins with Imidazole (for the former) or acidic elution (for the latter) followed by a desalting step (PD columns with Sephadex G25 resin, GE Healthcare) and if necessary, gel filtration chromatography (Superdex column, GE Healthcare) in PBS.

Example 2

Selective Binding to huNav1.7α.

Crude periplasmic extracts containing ISVDs from phage display selections (as described above) were screened in FACS for binding to huNav1.7α but not to huNav1.5a. Confirmatory binding FACS experiments with purified FLAG3-HIS6 tagged ISVD proteins revealed that the ISVDs all bind selectively to different stable cell lines expressing huNav1.7α and huNav157 chimera 14 (extracellular and transmembrane sequences of huNav1.7α, combined with intracellular sequences of huNav1.7α and huNav1.8α and the Navβ1, Navβ2, and Navβ3 subunits (see Table 8; FIG. 3A-FIG. 3I)), but not to cell lines expressing rhNav1.7α, huNav1.1α, huNav1.2α, huNav1.3α, huNav1.4α, huNav1.5α, huNav1.6α or huNav1.8α. For example, FIGS. 39A-39E show that F0103262CO2, F0103265B04, F0103275B05, F0103464B09, and F0103387G05 are specific for huNav1.7α with no binding to huNav1.1α, huNav1.2α, huNav1.3α, huNav1.4α, huNav1.5α, huNav1.6α or huNav1.8α. As used in Table 8, the drawings, and throughout the description, Navβ1, Navβ2, and Navβ3 are human homologs unless specifically identified otherwise.

TABLE 8

HEK

CHO
HEK
FlpIn

HEK
CHO

FlpIn
FlpIn
huNav
HEK293
FlpIn
FlpIn

huNav1.7α +
huNav1.7α +
157chimera14 +
huNav1.7α +
huNav1.5α +
rhNav1.7α +

SEQ
β1-β2- β3
β1-β2- β3
β1- β2-β3
β1
β1-β2- β3
β1-β2- β3

ID
(SEQ ID NO: 3)
(SEQ ID NO: 3)
(SEQ ID NO: 20)
(SEQ ID NO: 44)
(SEQ ID NO: 22)
(SEQ ID NO: 3)

ID #
NO:
pEC50 [M]
pEC50 [M]
pEC50 [M]
pEC50 [M]
pEC50 [M]
pEC50 [M]

F0103262B06
30
1.9E−08
2.5E−08
1.6E−08
2.0E−08
—
—

F0103262C02
31
3.2E−07
3.8E−07
4.7E−07
1.8E−07
—
—

F0103265A11
32
6.6E−08
3.7E−08
3.4E−08
8.4E−09
—
—

F0103265B04
33
5.4E−09
8.1E−09
8.5E−09
5.5E−09
—
—

F0103275B05
34
2.7E−08
3.7E−08
3.9E−08
2.8E−08
—
—

F0103362B08
35
1.1E−07
4.2E−08
ND
5.9E−08
—
—

F0103387G04
36
1.9E−08
1.6E−08
ND
6.7E−09
—
2.1E−07

F0103387G05
37
ND
3.1E−09
3.6E−09
1.2E−09
—
—

F0103345D07
38
2.2E−08
ND
ND
1.2E−08
—
—

F0103464B09
39
4.1E−09
ND
ND
4.1E−09
—
2.7E−08

Mean pEC50 ± standard deviation;

—, no binding observed;

ND, not determined

The amino acid sequences for the ten ISVDs (Nav1.7 binders) without the FLAG-HIS6 peptide (SEQ ID NO: 56) are shown in SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55, respectively.

Example 3

Affinity maturation was used to further improve the functional potencies of selected ISVDs by means of in vitro affinity maturation. In addition, as none of the selected ISVDs is cross-reactive to rhNav1.7α (with the exception of the weakly cross-reactive ISVD F0103387G04), the same process was applied to improve the NHP cross-reactivity to enable in vivo proof of concept (POC) studies in rhesus monkeys. In vitro affinity maturation of ISVDs is a two-stage process that aims to improve binding-related properties like affinity, species cross-reactivity or potency. First, all CDR-based residues are systematically changed to every possible amino acid on a one-by-one basis. The resulting libraries of single site substitution variants pooled per CDR are then screened for improvement of the desired property after which the hits are identified by means of Sanger sequencing. The beneficial single site substitutions are then combined into a library of combinatorial variants which are evaluated for further improvement of the desired property, followed by Sanger sequencing of hits. The generation the DNA fragments encoding the ISVD variants is either outsourced to commercial providers GeneWiz (South Plainfield, NJ) or IDT (Coralville, IA) or performed in house using commonly known molecular biology techniques such as site-directed mutagenesis, overlap extension PCR and oligonucleotide gene assembly (In Vitro Mutagenesis Protocols, 2^ndEdition (2002), Jeff Braman ed., Humana Press, Totowa NJ).

Affinity Maturation of F0103275B05 & F0103387G04

As ISVD F0103275B05 and rhNav1.7α cross-reactive F0103387G04 appear to be related ISVDs with highly similar CDRs (FIG. 4), it was decided to pursue these two ISVDs for affinity maturation in one and the same effort. A pooled single site saturation stage I library of F0103275B05 was constructed and crude periplasmic extracts of 2100 individual clones were prepared and screened in binding FACS to huNav1.7α and rhNav1.7α. Clones with a single mutation in CDR3, CDR2 or CDR1 residues showed an improved binding to rhNav1.7α, but much less so to huNav1.7α (FIG. 5).

The sequence analysis of 384 hits is summarized in Table 9. The stage I hits have substitutions in 7/10, 7/9, and 5/15 positions of respectively CDR1, CDR3 and CDR3. Interestingly, the substitutions in three of these positions (27, 28 and 53) recapitulate some of the differences between F0103275B05 and its rhNav1.7α cross-reactive relative F0103387G04 and thus bring additional confidence in the outcome of the stage I screening. These three substitutions were included in the design of the stage II combinatorial library (bottom row of Table 8), in which 11 positions were allowed to vary between the parental F0103275B05 and the highest ranked stage I hit residue. The stage II library thus captures 2¹¹=2048 different combinatorial variants.

TABLE 9

Summary sequence analysis of F0103275B05/F0103387G04 screening

stage I & design stage II affinity maturation libraries

CDR1
CDR3

Kabat #
26
27
28
29
30
31
32
33
34
35
50
51
52
53
54
55
56
57
58

′275B05
G
S
I
F
N
I
N
S
M
A
S
S
T
N
G
G
S
T
N

′387G04
G
P
V
F
N
I
N
K
M
A
S
V
T
P
T
G
S
I
S

Rank
1

P
V

L
W
S
R

R
Y

P
R
W
D
H
R

hits
2

W

W

L
R

R
W

stage I
3

V

V

4

Q

Stage II design
G
P
V
F
NL
IW
N
SR
M
AR
SY
S
T
P
G
GW
SD
TW
N

CDR3

Kabat #
93
94
95
96
97
98
99
100
100a
100b
100c
100d
100e
101
102

′275B05
N
A
L
L
Q
P
S
I
Y
D
I
S
R
T
Y

′387G04
N
A
L
L
Q
P
D
S
Y
S
N
T
R
T
Y

Rank
1
W
W

T

I

hits
2
D
W
E

K

stage I
3

G

F

4

Stage II design
NR
AW
L
L
Q
P
S
I
T
D
I
S
R
TI
Y

′274B05 = F0103275B05;

′387G04 = F0103387G04

Crude periplasmic of 2100 clones of the stage II combinatorial library were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. A large fraction of the variants displayed improved binding to rhNav1.7α compared to the huNav1.7α-selective parental F0103275B05 (FIG. 6), indicating that the library design successfully captured and improved the promise of the stage I library. No improvements for binding to huNav1.7α were observed for stage II, in line with the observations during stage I. The sequence analysis of 300 hits is summarized in Table 10. Compared to a randomly picked reference sample, the top 25% of the hits are enriched for the N93R substitution but display a lower proportion of the N30L, I31W, A35R, G55W and T57W substitutions. Compared to a randomly picked reference sample, the bottom 25% of the hits displayed a lower proportion of the I31W and A35R substitutions. An analysis of the subset of the top 25% hits that did not carry the N93R mutation revealed that these were enriched for the S33R, S50Y and S56D substitutions and had a lower proportion of A94W, compared to the reference sample.

TABLE 10

Summary sequence analysis of F0103275B05 screening stage II affinity maturation libraries

CDR1
CDR2
CDR3

Kabat #
N30L
I31W
S33R
A35R
S50Y
G55W
S56D
T57W
N93R
A94W
T101I

Reference
47%
44%
51%
39%
40%
43%
47%
47%
42%
53%
56%

sample

Top 25% of
26%
16%
40%
1%
53%
11%
46%
24%
77%
44%
47%

hits

Bottom 25%
43%
25%
46%
10%
51%
42%
48%
54%
46%
48%
42%

of hits

Top 25% of
31%
6%
88%
6%
63%
19%
56%
38%
NA
19%
50%

hits with N93

NA, not applicable

A number of combinatorial affinity maturation variants of F0103275B5 were then characterized in detail in binding FACS and electrophysiology (Table 11). All variants bound rhNav1.7α, many with greater affinity than F01033387G04. This was confirmed for most of them in 2-pulse (FIG. 7B) and single pulse (FIG. 7A) electrophysiology experiments. A subset of variants is equipotent on huNav1.7α and rhNav1.7α, with binding EC₅₀values of ±20 nM. The minimal number of mutations to a achieve this is four (S33R, S50Y, S56D and N93R) as exemplified by F010301461. F0103387G04 remains the best binder to huNav1.7α, most likely due to differences compared to F0103275B05 in other CDR positions. Variant F010300659 was the first variant with good rhNav1.7α cross-reactivity to be characterized, and as such was selected for in vivo assessment.

TABLE 11

Summary binding and functional characterization of F01033275B05 affinity variants

Part 1

Kabat # (mutations vs. F01033275B05)

ID #
S27
I28
I31
S33
S50
N53
G55
S56
T57
N93
A94
T101

F010300948
P
V
.
R
.
P
.
.
.
R
W
I

F010301462
P
V
.
R
Y
P
.
D
.
R
.
.

F010301459
P
V
.
R
.
P
.
D
.
R
.
.

F010301461
.
.
.
R
Y
.
.
D
.
R
.
.

F010300900
P
V
.
R
.
P
.
D
.
R
W
I

F010300880
P
V
.
R
.
P
.
D
.
R
W
.

F010301460
P
V
.
R
Y
P
.
.
.
R
.
.

F010300990
P
V
.
R
Y
P
.
.
.
.
.
I

F010301000
P
V
.
R
Y
P
.
D
.
.
.
I

F010300468
.
.
.
.
.
.
.
.
.
R
.
.

F010300796
P
V
.
R
Y
P
.
D
.
.
.
.

F010300631
P
V
.
.
Y
P
.
.
.
R
.
.

F010300684
P
V
W
.
.
P
.
.
.
R
.
I

F010300659
P
V
.
.
Y
P
W
D
W
R
W
.

F0103387G04
P
V
.
.
.
P
.
.
.
.
.
.

F010300477
.
.
.
.
.
.
.
.
.
.
W
.

F010300316
.
.
.
.
.
.
.
.
W
.
.
.

F0103275B05
.
.
.
.
.
.
.
.
.
.
.
.

Part 2

HEK

CHO FlpIn
HEKa/β1

rhNav1.7α + β1-

rhNav1.7α + β1-
(SEQ ID NO: 40)
HEKa
β2-β3
HEKa/β1

β2-β3
Nav1.7
huNav1.70α
(SEQ ID NO: 4)
(SEQ ID NO: 40)

(SEQ ID NO: 4)
(SEQ ID NO: 1)
(SEQ ID NO: 1)
single pulse
single pulse

ID #
EC50 [M]
EC50 [M]
EC50 [M]
IC50 [M]
IC50 [M]

F010300948
2.0E−08
6.8E−08
6.9E−08
5.8E−08
3.2E−07

F010301462
2.3E−08
2.2E−08
3.5E−08
ND
ND

F010301459
2.3E−08
1.8E−08
2.2E−08
ND
ND

F010301461
2.4E−08
2.1E−08
2.8E−08
ND
ND

F010300900
2.5E−08
7.0E−08
2.0E−08
3.6E−08
2.0E−07

F010300880
2.6E−08
4.4E−08
1.5E−08
1.2E−07
2.7E−08

F010301460
3.2E−08
2.6E−08
1.5E−08
ND
ND

F010300990
3.8E−08
6.5E−08
4.3E−09
1.0E−07
2.5E−07

F010301000
4.0E−08
6.0E−08
8.5E−09
ND
ND

F010300468
4.0E−08
ND
ND
ND
ND

F010300796
4.4E−08
3.8E−08
5.8E−09
ND
ND

F010300631
4.5E−08
ND
ND
1.7E−07
7.3E−08

F010300684
5.0E−08
ND
ND
2.1E−07
2.0E−07

F010300659
5.5E−08
2.7E−08
9.7E−09
1.8E−07
8.5E−08

F0103387G04
1.5E−07
6.7E−09
3.2E−09
ND
ND

F010300477
1.7E−07
ND
ND
1.2E−06
9.6E−08

F010300316
1.7E−07
ND
ND
ND
ND

F0103275B05
—
4.8E−08
1.0E−08
ND
ND

Part 3

huNav1.7α

rhNav1.7α

(SEQ ID NO: 1)

(SEQ ID NO: 2)

2-pulse IC50 [M]

2-pulse IC50 [M]

ID #
P1
P2
P1
P2

F010300948
ND
ND
ND
ND

F010301462
ND
ND
ND
ND

F010301459
ND
ND
ND
ND

F010301461
ND
ND
ND
ND

F010300900
ND
ND
ND
ND

F010300880
ND
ND
ND
ND

F010301460
ND
ND
ND
ND

F010300990
ND
ND
ND
ND

F010301000
ND
ND
ND
ND

F010300468
5.0E−06
4.0E−06
6.0E−06
3.0E−06

F010300796
ND
ND
ND
ND

F010300631
1.0E−06
8.0E−07
2.0E−06
8.0E−07

F010300684
2.0E−06
2.0E−06
7.0E−06
1.0E−06

F010300659
1.0E−06
8.0E−07
1.0E−06
7.0E−07

F0103387G04
7.0E−07
4.0E−07
7.0E−06
3.0E−06

F010300477
2.0E−06
2.0E−06
3.0E−06
1.0E−06

F010300316
2.0E−06
1.0E−06
5.0E−06
4.0E−06

F0103275B05
ND
ND
ND
ND

—, no binding detected; ND, not determined

Affinity Maturation of F01033265A11

A pooled single site saturation library of F0103265A11 was constructed and crude periplasmic extracts of 1848 individual clones were prepared and screened in binding FACS on huNav1.7a and rhNav1.7α. Clones with a single mutation in CDR2, CDR3 or CDR1 residues showed an improved binding to huNav1.7α, but not to rhNav1.7α (FIG. 12).

The sequence analysis of 288 hits is summarized in Table 12. The stage I hits have substitutions in 3 of 10, 7 of 11, and 4 of 6 positions of respectively CDR1, CDR3 and CDR3. Of interest, four CDR2 positions (51, 53, 56 and 57) have substitutions to a Trp residue. The stage II library design captures 2 11=2048 different combinatorial variants.

TABLE 12

Summary sequence analysis of F0103265A11 screening stage

I & design stage II affinity maturation libraries

CDR1
CDR2

Kabat #
26
27
28
29
30
31
32
33
34
35
50
51
52
53
54

F0103265A11
G
M
L
F
N
A
N
T
Q
G
F
I
F
S
G

Rank
1

K

Y
R

W

W

hits
2

L

stage I
3

R

4

H

5

F

Stage II design
G
M
L
F
NY
AR
N
T
Q
G
F
IW
F
SW
G

CDR2
CDR3

Kabat #
55
56
57
58
59
60
93
94
95
101
102
103

F0103265A11
G
Y
T
N
Y
V
S
L
S
R
Y
L

Rank
1
M
W
R
T

N
A
A

V

Q

hits
2
N

V
S

T

L

stage I
3

W
A

T

4

L

5

Stage II design
G
YW
TV
NT
Y
VN
SA
L
S
RV
Y
LQ

Crude periplasmic of 2016 clones of the stage II combinatorial library were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. A large fraction of the variants displayed improved binding to huNav1.7α compared to the parental F0103265A11 (FIG. 9), indicating that the library design successfully captured and improved the promise of the stage I library. No improvements for binding to rhNav1.7α were observed for stage II, in line with the observations during stage I. The sequence analysis of 288 hits is summarized in Table 13. Compared to a randomly picked reference sample, the top 25% of the hits are enriched for the A31R, V60N and S93A substitutions but display a lower proportion of the N30Y, 151W, S53W, T57V and N58T substitutions. Compared to a randomly picked reference sample, the bottom 25% of the hits are enriched for the T57V, S93A and L103Q substitutions but display a lower proportion of the N30Y, I51W and S53W substitutions.

TABLE 13

Summary sequence analysis of F0103265A11 screening stage II affinity maturation libraries

CDR1
CDR2
CDR3

Kabat #
N30Y
A31R
I51W
S53W
Y56W
T57V
N58T
V60N
S93A
R101V
L103Q

Reference
22%
30%
27%
37%
30%
45%
55%
37%
38%
18%
55%

sample

Top 25% of
11%
51%
11%
17%
27%
37%
41%
79%
56%
14%
60%

hits

Bottom 25% of
11%
30%
11%
25%
25%
56%
51%
41%
62%
27%
68%

hits

A number of combinatorial affinity maturation variants of F0103265A11 were then characterized in detail in binding FACS and electrophysiology (Table 14). Most variants displayed clear improvements in binding EC50 and Bmax values on huNav1.7α, compared to parental F0103265A11. This became even more pronounced when huNav1.7α was expressed in the absence of Navβ-subunits: no binding was observed for parental 265A11, whereas many affinity maturation variants showed clear binding curves to the HEKa-only line. The previously observed β-subunit dependency of F0103265A11 was improved by the affinity maturation process. Clear improvements in functional inhibition of the ion channel were observed (last column of Table 14), compared to the marginal functional inhibition observed in the past for parental F0103265A11.

TABLE 14

Summary binding and functional characterization of F0103265A11 affinity variants

Part 1

Kabat # (mutations vs. F01033265A11)

ID #
N30
A31
I51
S53
Y56
T57
N58
V60
S93
R101
L103

F010301458
.
R
.
.
W
.
.
N
A
.
Q

F010301463
.
R
.
.
.
.
.
N
A
.
Q

F010301080
.
.
.
W
.
V
T
N
.
.
.

F010301129
.
.
.
.
W
.
.
N
A
.
Q

F010301162
.
R
.
.
W
.
.
N
.
.
Q

F010301191
.
R
.
.
W
.
.
N
A
V
Q

F010301055
.
.
.
W
.
V
.
.
.
.
.

F010301139
.
R
W
.
.
V
.
N
.
.
Q

F010301090
.
.
.
.
W
.
T
N
.
.
Q

F010301188
.
R
.
.
.
.
.
N
.
.
Q

F010301175
.
R
.
.
.
.
T
.
.
.
.

F010301111
.
.
.
.
.
V
.
N
A
.
.

F010301059
.
.
.
.
.
V
.
N
A
.
Q

F010300535
.
.
.
W
.
.
.
.
.
.
.

F010301126
.
R
.
.
.
V
T
N
.
.
.

F010301113
.
R
.
.
.
.
.
N
.
V
Q

F010301099
.
.
.
.
W
V
T
.
.
.
.

F010300536
.
.
.
.
.
.
T
.
.
.
.

F010301232
.
R
.
.
.
.
T
.
A
.
Q

F010301138
Y
.
.
.
.
.
.
N
A
.
Q

F010300534
.
.
.
.
.
V
.
.
.
.
.

F0103265A11
.
.
.
.
.
.
.
.
.
.
.

Part 2

HEKa/β1

(SEQ ID

HEK FlpIn
NO: 40)

HEKa

Nav1.7α + β1-
huNav1.7α

Nav1.7α
HEKa/β1

β2-β3
(SEQ ID
Bmax
(SEQ ID
(SEQ ID

(SEQ ID NO:
NO: 1)
relative
NO: 1)
NO: 40)

ID #
3) EC50 [M]
EC50 [M]
to ′535
EC50 [M]
IC50 [M]

F010301458
4.1E−09
2.2E−09
120%
8.6E−09
ND

F010301463
4.3E−09
2.5E−09
120%
2.9E−08
ND

F010301080
5.1E−09
3.0E−09
122%
2.6E−08
1.4E−08

F010301129
ND
3.6E−09
103%
ND
1.3E−07

F010301162
ND
3.8E−09
115%
ND
3.2E−08

F010301191
ND
4.1E−09
119%
ND
6.3E−08

F010301055
ND
4.4E−09
117%
ND
ND

F010301139
ND
4.7E−09
117%
ND
ND

F010301090
ND
4.8E−09
102%
ND
2.2E−08

F010301188
ND
5.1E−09
104%
ND
ND

F010301175
ND
5.4E−09
111%
ND
ND

F010301111
ND
5.4E−09
99%
ND
ND

F010301059
ND
5.4E−09
99%
ND
ND

F010300535
1.6E−08
5.5E−09
100%
7.2E−07
ND

F010301126
ND
5.6E−09
118%
ND
ND

F010301113
ND
5.7E−09
111%
ND
ND

F010301099
ND
7.4E−09
87%
ND
ND

F010300536
2.7E−08
9.0E−09
77%
6.6E−07
ND

F010301232
ND
9.3E−09
112%
ND
ND

F010301138
ND
9.7E−09
107%
ND
ND

F010300534
7.2E−08
1.1E−08
62%
—
ND

F0103265A11
7.7E−08
1.1E−08
34%
—
ND

—, no binding detected;

ND, not determined

Affinity Maturation of F0103265B04

A pooled single site saturation library of F0103265B04 was constructed and crude periplasmic extracts of 2016 individual clones were prepared and screened in binding FACS on huNav1.7a and rhNav1.7α. No clones with a single mutation in CDR3, CDR2 or CDR1 residues showed an improved binding to huNav1.7a or rhNav1.7α (FIG. 10). The outliers in the top right quadrant of FIG. 10 was determined to be a contamination with F0103240B04, a 132 binding ISVD.

Affinity Maturation of F0103387G05

A pooled single site saturation library of F0103387G05 was constructed and crude periplasmic extracts of 3360 individual clones were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. Clones with a single mutation of CDR2, CDR3 or CDR1 residues showed weakly improved binding to huNav1.7α, but not to rhNav1.7α (FIG. 11).

Sequence analysis of 384 hits revealed an enrichment for certain positions and mutations, but as there were no outspoken improvements in binding observed, it was decided to first characterize a number of stage I variants rather than combining these in a stage II combinatorial library. Binding FACS experiments (Table 15) revealed that most of the tested variants were comparable to parental F0103387G05. Interestingly, a number of CDR1- and CDR2-based (Kabat positions 23, 53, 54 and 58) mutations, all substitutions of Asp with Gly, displayed subtle improvements compared to parental F0103387G05. Combinations of these substitutions further improved the binding in a subtle way (Table 15). Combinations of these substitutions further improved the binding in a subtle way with D23A and D58G substitutions contributing the most (Table 15), resulting in the selection of F0103301563 as the preferred variant.

TABLE 15

Summary binding characterization of F0103387G05 affinity variants

CHO FlpIn
HEKa/β1

huNav1.7α +
(SEQ ID NO: 40)
HEKa

β1-β2-β3
huNav1.7α
huNav1.7α

(SEQ ID NO: 3)
(SEQ ID NO: 1)
(SEQ ID NO: 1)

ID #
Mutations vs. 387G05
EC50 [M]
EC50 [M]
EC50 [M]

F010301559
D23A, D54G, D58G
1.8E−09
9.2E−10
8.7E−10

F010301558
D23A, D53G, D58G
1.8E−09
9.3E−10
1.5E−09

F010301563
D23A, D58G
1.9E−09
1.0E−09
1.5E−09

F010301560
D53G, D54G, D58G
2.1E−09
1.1E−09
1.2E−09

F010301565
D53G, D58G
2.1E−09
1.0E−09
1.4E−09

F010301566
D54G, D58G
2.2E−09
1.0E−09
1.2E−09

F010301556
D23A, D53G, D54G, D58G
2.3E−09
1.4E−09
1.8E−09

F010301561
D23A, D53G
2.5E−09
1.2E−09
2.2E−09

F010301562
D23A, D54G
2.5E−09
1.9E−09
1.2E−09

F010301346
D58G
3.4E−09
1.6E−09
1.5E−09

F010301564
D53G, D54G
3.5E−09
2.4E−09
1.9E−09

F010301350
D58V
3.5E−09
1.7E−09
1.6E−09

F010301314
D23A
3.5E−09
1.8E−09
1.9E−09

F010301557
D23A, D53G, D54G
3.8E−09
2.1E−09
2.0E−09

F010301367
D53G
4.2E−09
1.9E−09
2.2E−09

F010301344
D54G
4.2E−09
2.1E−09
1.3E−09

F010301372
S100dG
4.5E−09
2.3E−09
2.5E−09

F010301445
S100dY
4.6E−09
2.0E−09
2.3E−09

F0103387G05

4.7E−09
1.7E−09
2.7E−09

F010301418
Y102W
4.9E−09
ND
ND

F010301328
T57V
5.0E−09
ND
ND

F010301409
H100fV
5.1E−09
ND
ND

F010301387
V100A
5.2E−09
ND
ND

F010301360
A56T
5.2E−09
ND
ND

F010301309
L29V
5.4E−09
ND
ND

F010301313
R35K
5.5E−09
ND
ND

F010301304
I31W
5.7E−09
ND
ND

F010301440
G100hT
5.7E−09
ND
ND

F010301301
I31T
7.3E−09
ND
ND

F010301335
R50V
7.9E−09
ND
ND

F010301425
G100bV
1.5E−08
ND
ND

F010301416
T101Q
—
ND
ND

—, no binding detected;

ND, not determined

Affinity Maturation of F0103362B08

A pooled single site saturation library of F0103362B08 was constructed and crude periplasmic extracts of 4032 individual clones were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. Clones with a single mutation of CDR2, CDR3 or CDR1 residues showed weakly improved binding to huNav1.7α, but not to rhNav1.7α (FIG. 12).

Sequence analysis of 326 hits revealed an enrichment for certain positions and mutations, but as there were no outspoken improvements in binding observed, it was decided to first characterize a number of stage I variants rather than combining these in a stage II combinatorial library. Binding FACS experiments (Table 16) revealed that most of the tested variants were comparable to parental F0103362B8. A number of mutations (Kabat positions 50, 97, 99 and 1000, consistently displayed subtle improvements compared to parental 362B08 across two different huNav1.7α cell lines.

TABLE 16

Summary binding characterization of F0103362B08 affinity variants

CHO FlpIn
HEKa/β1

huNav1.7α +
(SEQ ID NO: 40)
HEKa

β1-β2-β3
huNav1.7α
huNav1.7α

(SEQ ID NO: 3)
(SEQ ID NO: 1)
(SEQ ID NO: 1)

ID #
Mutations vs. 362B08
EC50 [M]
EC50 [M]
EC50 [M]

F010301595
D97A
6.2E−08
1.7E−08
ND

F010301606
Y100fK
6.5E−08
2.4E−08
ND

F010301627
G50A
7.6E−08
2.3E−08
ND

F010301598
G99Q
8.9E−08
2.9E−08
ND

F010301607
Y100fA
9.0E−08
3.0E−08
ND

F010301591
G99K
9.3E−08
4.3E−08
ND

F010301574
G35A
1.0E−07
2.9E−08
ND

F010301594
R100aK
1.1E−07
2.9E−08
ND

F010301584
W52aT
1.1E−07
4.2E−08
ND

F010301593
R96K
1.1E−07
4.0E−08
ND

F010301585
V56P
1.1E−07
5.3E−08
ND

F010301567
F29Y
1.1E−07
2.4E−08
ND

F0103362B08

1.1E−07
3.4E−08
1.9E−08

F010301578
S30G
1.2E−07
3.0E−08
ND

F010301629
G50S
1.2E−07
4.2E−08
ND

F010301589
I55P
1.2E−07
4.1E−08
ND

F010301579
R27K
1.3E−07
3.6E−08
ND

F010301596
G99A
1.4E−07
5.0E−08
ND

F010301621
Y100fG
1.5E−07
3.4E−08
ND

F010301617
F100cQ
1.7E−07
4.4E−08
ND

F010301586
W52aA
1.7E−07
5.1E−08
ND

F010301612
Y100fT
1.7E−07
4.4E−08
ND

F010301622
Y100fD
1.7E−07
4.0E−08
ND

F010301580
R27T
1.8E−07
3.5E−08
ND

F010301619
F100cK
1.8E−07
4.7E−08
ND

F010301618
Y102H
1.9E−07
3.9E−08
ND

F010301609
Y100fQ
1.9E−07
3.7E−08
ND

F010301604
R100aQ
1.9E−07
5.2E−08
ND

F010301592
R100aS
2.0E−07
6.7E−08
ND

F010301568
G35T
3.1E−07
1.1E−07
ND

F010301657
A14P, D97A, G99Q, Y100fK
ND
1.6E−08
2.0E−08

F010301658
A14P, G50A, D97A, G99Q, Y100fK
ND
2.0E−08
2.3E−08

F010301659
A14P, G50A, D97A
ND
1.2E−08
1.1E−08

F010301661
A14P, G50A, Y100fK
ND
1.8E−08
1.6E−08

F010301662
A14P, D97A, G99Q
ND
1.7E−08
1.9E−08

F010301663
A14P, D97A, Y100fK
ND
1.4E−08
1.3E−08

F010301664
A14P, G99Q, Y100fK
ND
1.8E−08
1.3E−08

F010301665
A14P, G50A, D97A, Y100fK
ND
1.1E−08
8.8E−09

F010301666
A14P, G50A, G99Q, Y100fK
ND
1.4E−08
1.0E−08

ND, not determined

Affinity Maturation of F0103464B09

A pooled single site saturation library of F0103464B09 was constructed and crude periplasmic extracts of 3356 individual clones were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. Clones with a single mutation of mainly CDR2 residues showed weakly improved binding to rhNav1.7α, but hardly not to huNav1.7α (FIG. 13).

Sequence analysis of 186 hits revealed an enrichment for certain positions and mutations, particularly in CDR1 and CDR2. It was decided to first characterize a number of stage I variants based on their sequence enrichment in the hits and/or improved binding vs. parental controls (Table 17). Compared to parental F0103464B09, a number of the tested substitutions clearly improved binding to rhNav1.7α in terms of Bmax while being neutral for binding to huNav1.7α.

TABLE 17

Summary binding characterization of F0103464B09 affinity variants

CHO FlpIn
CHO FlpIn
CHO FlpIn

rhNav1.7α +
rhNav1.7α +
huNav1.7α +

β1-β2-β3
β1-β2-β3
β1-β2-β3

Mutations vs.
(SEQ ID NO: 4)
(SEQ ID NO: 4)
(SEQ ID NO: 3)

ID #
F103464B09
EC50 [M]
Bmax
EC50 [M]

F010301892
V33L
9.1E−09
100%
4.E−09

F010301885
N53E
1.8E−08
100%
8.0E−09

F010301881
G54W
5.8E−09
99%
4.2E−09

F010301888
S26H
8.7E−09
95%
5.3E−09

F010301889
S95A
8.5E−09
92%
5.4E−09

F010301878
G54E
2.7E−08
83%
7.1E−09

F010301886
N58Q
1.5E−08
81%
4.3E−09

F010301867
A28Q
2.2E−08
74%
4.8E−09

F010301880
G54S
2.4E−08
73%
4.9E−09

F010301890
T35V
2.0E−08
58%
5.0E−09

F0103464B09

3.3E−08
54%
5.4E−09

F010301887
R31Q
5.9E−08
51%
7.5E−09

F010301883
I51V
3.4E−08
48%
4.8E−09

F010301884
N53A
5.0E−08
47%
6.0E−09

F010301891
T57V
2.6E−08
45%
4.7E−09

F010301882
I30V
4.1E−08
43%
4.5E−09

F010301879
G54Q
ND
ND
ND

ND, not determined

Based on these observations, a combinatorial library was generated with a diversity of 320 different variants, as summarized by Table 17. Crude periplasmic of 2880 clones of the stage II combinatorial library were prepared and screened in binding FACS on huNav1.7α and rhNav1.7α. A large fraction of the variants display improved binding to rhNav1.7α compared to the parental F0103464B09 (FIG. 14), indicating that the library design successfully captured and improved the promise of the stage I library. No outspoken improvements for binding to huNav1.7α were observed for stage II, in line with the observations during stage I.

The sequence analysis of 273 hits (per 96-well plate, each time top three hits on huNav1.7α and top seven hits on rhNav1.7α) is summarized in Table 18. Compared to a randomly picked reference sample, the V33L, G54W and S95A substitutions are underrepresented in the top three hits on huNav1.7α and rhNav1.7α. As such, the variants with these substitutions were excluded from further analysis. Furthermore, 38/96 (40%) of the top 3 hits on huNav1.7α matched the parental F0103464B09 sequence, again suggesting that no major improvements on huNav1.7α could be expected from this library. As no outspoken sequence enrichments could be observed from Table 18, the following criteria were applied to further narrow down the number of variants for detailed characterization:

- sequenced multiple times and at least once present in both top 2 hits on huNav1.7α and rhNav1.7α;
- no deamidation motif on position 53;
- less than 5 mutations compared to parental F0103464B09.
  
  The resulting combinatorial variants (Table 19) were supplemented with one variant carrying V33L substitution, as this is one of the most promising single substitutions (Table 17). These variants were combined with a two variable sequence optimization substitutions R39Q and A63V in a background containing a large number of sequence optimization substitutions (L11V, T24A, T25S, V40A, E44Q, F62S, S68T, M77T, T79Y, R81Q, S82aN, N82bS, K83R, G88A, V89L, N99S).

TABLE 18

Summary sequence analysis of F103464B09 screening stage II affinity maturation libraries

CDR1
CDR2
CDR3

Kabat #
S26H
A28Q
V33L
N53E
G54W
G54E
G54S
G54Q
N58Q
S95A

Reference
55%
41%
48%
48%
10%
24%
28%
21%
72%
52%

sample

Top 3 hits
47%
58%
4%
42%
0%
20%
29%
11%
62%
9%

on huNav1.7

Top 3 hits
51%
58%
19%
31%
1%
21%
19%
15%
85%
36%

on rhNav1.7

TABLE 19

Summary of selected F103464B09 affinity variants

Kabat # (mutations vs. F103464B09)

ID
S26
A28
V33
N53
G54
N58

A28Q G54E
.
Q
.
.
E
.

A28Q G54E N58Q
.
Q
.
.
E
Q

A28Q N53E G54S N58Q
.
Q
.
E
S
Q

S26H A28Q G54E N58Q
H
Q
.
.
E
Q

S26H A28Q N53E N58Q
H
Q
.
E
.
Q

S26H N53E G54S N58Q
H
.
.
E
S
Q

S26H N53E N58Q
H
.
.
E
.
Q

S26H V33L N53E G54S
H
.
L
E
S
.

In the course of the F0103464B09 sequence optimization process subtle drops in binding to rhNav1.7α were observed for the following substitutions: R39Q, A63V, T79Y, R81Q, and N99S. R39Q substitution also resulted in a subtle drop in binding to huNav1.7α. The combination of these, as present in the background in which the combinatorial variation was introduced, resulted in the complete abolishment of binding to rhNav1.7α for the controls that do not carry any of the affinity maturation substitutions (F010302365, F010302366 and F010302368 in Table 20) and the same was observed for the variants combining the A28Q G54E substitutions. Less outspoken, none of the variants combining the A28Q G54E N58Q, S26H A28Q G54E N58Q, or A28Q N53E G54S N58Q substitutions reached maximum binding levels to rhNav1.7α (Table 20). A similar observation was made for the variants combining the S26H V33L N53E G54S substitutions, which also resulted in a drop in binding EC50 to huNav1.7α. The three remaining combinations S26H N53E N58Q, S26H N53E G54S N58Q and S26H A28Q N53E N58Q were highly comparable for their binding to huNav1.7α and rhNav1.7α (Table 20). The S26H N53E N58Q combination was then selected as it achieves the same binding improvements with one mutation less than the two others.

TABLE 20

Summary binding characterization of F0103464B09 combinatorial

affinity and sequence optimization variants

Part 1

Substitutions vs. F103464B09 (L11V, T24A, T25S, V40A, E44Q, F62S,

S68T, M77T, T79Y, R81Q, S82aN, N82bS, K83R, G88A, V89L, N99S)

ID #
S26
A28
V33
R39
N53
G54
N58
A63

F010302365
.
.
.
.
.
.
.
.

F010302368
.
.
.
Q
.
.
.
V

F010302366
.
.
.
Q
.
.
.
.

F010302341
.
Q
.
Q
.
E
.
.

F010302357
.
Q
.
Q
.
E
.
V

F010302333
.
Q
.
.
.
E
.
.

F010302349
.
Q
.
.
.
E
.
V

F010302364
H
.
L
Q
E
S
.
V

F010302348
H
.
L
Q
E
S
.
.

F010302356
H
.
L
.
E
S
.
V

F010302340
H
.
L
.
E
S
.
.

F010302339
H
.
.
.
E
.
Q
.

F010302347
H
.
.
Q
E
.
Q
.

F010302355
H
.
.
.
E
.
Q
V

F010302363
H
.
.
Q
E
.
Q
V

F010302337
H
Q
.
.
E
.
Q
.

F010302345
H
Q
.
Q
E
.
Q
.

F010302353
H
Q
.
.
E
.
Q
V

F010302361
H
Q
.
Q
E
.
Q
V

F010302334
.
Q
.
.
.
E
Q
.

F010302342
.
Q
.
Q
.
E
Q
.

F010302350
.
Q
.
.
.
E
Q
V

F010302358
.
Q
.
Q
.
E
Q
V

F010302336
H
Q
.
.
.
E
Q
.

F010302344
H
Q
.
Q
.
E
Q
.

F010302352
H
Q
.
.
.
E
Q
V

F010302360
H
Q
.
Q
.
E
Q
V

F010302338
H
.
.
.
E
S
Q
.

F010302346
H
.
.
Q
E
S
Q
.

F010302354
H
.
.
.
E
S
Q
V

F010302362
H
.
.
Q
E
S
Q
V

F010302335
.
Q
.
.
E
S
Q
.

F010302343
.
Q
.
Q
E
S
Q
.

F010302351
.
Q
.
.
E
S
Q
V

F010302359
.
Q
.
Q
E
S
Q
V

Part 2

CHO FlpIn
CHO FlpIn

CHO FlpIn

rhNav1.7α +
rhNav1.7α +

huNav1.7α +
CHO FlpIn
β1- β2-β3
β1- β2-β3

β1-β2-β3 (SEQ
huNav1.7α + β1-
(SEQ ID
(SEQ ID

ID NO: 3)
β2-β3 (SEQ ID
NO: 4)
NO: 4)

ID #
EC50 [M]
NO: 3) Bmax
EC50 [M]
Bmax

F010302365
3.1E−09
100%
—
—

F010302368
3.9E−09
100%
—
—

F010302366
3.7E−09
100%
—
—

F010302341
5.3E−09
100%
—
—

F010302357
5.3E−09
100%
—
—

F010302333
4.1E−09
100%
—
—

F010302349
4.4E−09
100%
—
—

F010302364
7.2E−09
100%
1.7E−08
74%

F010302348
6.7E−09
100%
1.4E−08
95%

F010302356
5.3E−09
100%
1.2E−08
81%

F010302340
4.6E−09
100%
1.3E−08
96%

F010302339
4.3E−09
100%
6.8E−09
100%

F010302347
4.6E−09
100%
9.6E−09
100%

F010302355
3.5E−09
100%
6.8E−09
97%

F010302363
4.2E−09
100%
9.3E−09
96%

F010302337
3.9E−09
100%
6.8E−09
100%

F010302345
4.9E−09
100%
1.2E−08
96%

F010302353
4.2E−09
100%
7.9E−09
93%

F010302361
4.8E−09
100%
1.1E−08
92%

F010302334
4.0E−09
100%
1.1E−08
70%

F010302342
4.0E−09
100%
1.6E−08
61%

F010302350
3.5E−09
100%
1.5E−08
46%

F010302358
4.5E−09
100%
3.7E−08
37%

F010302336
3.6E−09
100%
9.7E−09
79%

F010302344
4.3E−09
100%
1.3E−08
78%

F010302352
3.6E−09
100%
1.1E−08
60%

F010302360
3.5E−09
100%
1.5E−08
58%

F010302338
3.4E−09
100%
7.9E−09
100%

F010302346
5.0E−09
100%
1.2E−08
99%

F010302354
4.2E−09
100%
7.9E−09
96%

F010302362
5.4E−09
100%
1.4E−08
89%

F010302335
4.1E−09
100%
1.3E−08
84%

F010302343
6.4E−09
100%
1.8E−08
84%

F010302351
5.6E−09
100%
1.5E−08
72%

F010302359
6.4E−09
100%
2.4E−08
66%

Example 4

Competitive Binding to huNav1.7α

Competition FACS assays were performed with CMYC3-tagged ISVD F0103265B04 or F0103275B05(N93R) affinity maturation variant on a HEK FlpIn huNav1.7α+β1−β2−β3 transgenic cell line. Briefly, cells were resuspended in FACS buffer (PBS, 2% FBS, 0.05% NaN₃) and 1×10⁵cells/well were transferred to 96-well V-bottom plates. Cells were subsequently resuspended in a 100 μL mixture of purified ISVD (dilution series) and CMYC3-tagged ISVD F0103265B04 (at a concentration equivalent to EC30) followed by incubation for 1.5 hours at 4° C. Residual binding of CMYC3-tagged ISVD F0103265B04 was detected with 1004 murine anti-CMYC (1/250 dilution) (Bio-Rad, catalog #MCA2200) followed by PE-conjugated goat anti-murine (Jackson Immunoresearch, catalog #115-116-071). Between each step, the cells were centrifuged for 5 minutes at 200 g and washed with 100 μL/well FACS buffer. Prior to the read-out, the samples were resuspended in 5 nM TOPRO3 (Molecular probes, catalog #T3605) to exclude dead cells. F0103262CO2, F0103262B06, F0103265A11, F0103265B04, F0103275B05, F0103362B08, and F0103387G04 all compete with F0103265B04 for binding to huNav1.7α, in contrast to an irrelevant control ISVD (IRR) (see Table 21 and FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D). The data shown in the figures and summarized in Table 20 suggests that all extracellular anti-Nav1.7α leads bind to an overlapping footprint.

TABLE 21

HEK FlpIn
CHO FlpIn
CHO FlpIn

huNav1.7α +
huNav1.7α +
rhNav1.7α +

β1-β2-β3
β1-β2-β3
β1-β2-β3

(SEQ ID NO: 3)
(SEQ ID NO: 3)
(SEQ ID NO: 4)

vs. EC30 of
vs. EC25 of
vs. EC40 of

F103265B04
F103275B05(N93R)
F103275B05(N93R)

ID #
IC50 [M]
IC50 [M]
IC50 [M]

F0103262C02
5.3E−07
ND
ND

F0103262B06
4.9E−08
ND
ND

F0103275B05
9.8E−08
ND
ND

F0103265B04
1.8E−08
ND
ND

F0103265A11
7.5E−08
ND
ND

F0103362B08
1.5E−07
ND
ND

F0103387G05
9.4E−09
1.0E−08
—

F0103387G04
5.9E−08
ND
ND

F0103464B09
ND
1.2E−08
7.9E−08

F0103454D07
ND
5.8E−08
—

ND, not determined

Example 5

Binding to huNav1.7α-Nav1.5 Chimeras

FACS binding studies (as described above) were performed on HEK293T cells transiently transfected with expression vectors encoding a huNav1.7α or rhNav1.7α fused at the C-terminus via a P2A viral peptide linker to a single polypeptide encoding sodium channel beta subunits Navβ1, Navβ2, and Navβ3 in tandem (β1-β2-β3; SEQ ID NO:21). Similarly, HEK293T cells transiently transfected with expression vectors encoding chimeric variants of huNav1.7α in which individual domains are replaced by their huNav1.5α counterparts (chimeras 1 to 4 in FIG. 16) or with chimeric variants of huNav1.5 in which individual domains are replaced by their huNav1.7α counterparts (chimeras 5 to 8 in FIG. 16) fused at the C-terminus via a P2A viral peptide linker to β1-β2-β3 as above. See Table 7 for description of the expression vectors encoding the chimeras, Table 21 and FIG. 16).

From experiments summarized in Table 22 and shown in FIG. 17A-FIG. 17C and FIG. 18A-18C, it could be concluded that DI of huNav1.7α is necessary and sufficient for the binding of F0103262CO2, F0103262B06, F0103265B04, F0103275B05, and F0103265A11 (see FIG. 16). From a separate experiment (Table 22 and FIGS. 19A-19B) with a chimeric variant of huNav1.7α in which the DI S5-S6 sequence is replaced by the huNav1.5 counterpart (chimera 12 in FIG. 16), it could be concluded that DI S5-S6 is necessary for the binding of F0103262CO2, F0103262B06, F0103265B04, F0103275B05, and F0103265A11 to huNav1.7α. In addition, F0103275B05 appears to also interact with the adjoining DIV VSD (Table 22, FIGS. 17A-17C, FIGS. 18A-18C, and FIGS. 20A-20B). Control antibodies murine anti-Nav1.7α mAb S68-6 (Abcam, catalog #ab85015) and rabbit anti-Nav1.5α pAb (Alomone Labs, catalog #ASC-013) recognize an epitope at the intracellular C-terminal part of their respective channel.

TABLE 22

SEQ

ID
DI
DII
DIII
DIV

ID #
NO:
S1-S2
S3-S4
S5-S6
S1-S2
S3-S4
S5-S6
S1-S2
S3-S4
S5-S6
S1-S2

huNav1.7α + β1-
3

β2-β3

huNav157chim1 +
10

X

β1-β2-β3

huNav157chim2 +
11

X
X
X

β1-β2-β3

huNav157chim3 +
12

X
X
X

β1-β2-β3

huNav157chim4+
13
X
X
X

β1-β2-β3

huNav157chim5+
14
X
X
X
X
X
X
X
X
X

β1-β2-β3

huNav157chim6+
15
X
X
X
X
X
X

X

β1-β2-β3

huNav157chim7+
16
X
X
X

X
X
X
X

β1-β2-β3

huNav157chim8+
17

X
X
X
X
X
X
X

β1-β2-β3

huNav157chim9+
18

β1-β2-β3

huNav157chim12
19

X

+β1-β2-β3

huNav157chim18
29

X

+β1-β2-β3

huNav157chim22
21

X

+β1-β2-β3

DIV

ID #
S3-S4
S5-S6
F0103262C02
F0103275B05
F0103265B04
F0103262B06
F0103265A11

huNav1.7α + β1-

+
+
+
+
±

β2-β3

huNav157chim1 +
X
X
+
−
+
+
±

β1-β2-β3

huNav157chim2 +

+
+
+
+
±

β1-β2-β3

huNav157chim3 +

+
+
+
+
±

β1-β2-β3

huNav157chim4+

−
−
−
−
−

β1-β2-β3

huNav157chim5+

−
−
−
−
−

β1-β2-β3

huNav157chim6+
X
X
−
−
−
−
−

β1-β2-β3

huNav157chim7+
X
X
−
−
−
−
−

β1-β2-β3

huNav157chim8+
X
X
+
±
+
+
±

β1-β2-β3

huNav157chim9+

X
+
+
+
+
±

β1-β2-β3

huNav157chim12

−
−
−
−
−

+β1-β2-β3

huNav157chim18
X

+
±
+
+
±

+β1-β2-β3

huNav157chim22

+
±
+
+
±

+β1-β2-β3

X boxes, Nav1.5;

empty boxes, Nav1.7α;

+, strong binding;

± weak binding;

−, no binding

Example 6

Binding to huNav1.7α-rhNav1.7α Chimeras

FACS binding studies (as described above) were performed on HEK293T cells transiently transfected with a chimeric variant of huNav1.7α in which all the huNav1.7α-rhNav1.7α polymorphisms of DI are present (N146S, V1941, F276V, R277Q, E281V, V331M, E504D, D507E, S508N, N533S). Replacing the huNav1.7α DI sequence for that of rhNav1.7α is sufficient to abolish the binding of F0103262CO2, F0103265B04, F0103262B06, and F0103265A11 to huNav1.7α, recapitulating the absence of binding on rhNav1.7α (see FIGS. 21A-21B).

Based on the huNav1.7α model (as described above) the following huNav1.7α-rhNav1.7α polymorphisms can be allocated to the extracellular part of DI: N146S, F276V, R277Q, E281V and V331M. The first of the residues is in DI S1-S2 whereas the latter four residues belong to DI S5-S6. FACS binding studies (as described above) were performed to stable CHO FlpIn cell lines expressing different variants of huNav1.7+β1−β2−β3 each including one of the four possible extracellular huNav1.7α-rhNav1.7α polymorphisms in the DI S5-S6 region: F276V, R277Q, E281V and V331M (Table 23 and FIGS. 22A-22F). Individual polymorphisms were each sufficient to abolish the binding of F0103265B04, F0103362B08, F010301080, and F0103262B06 to huNav1.7α, recapitulating the absence of binding on rhNav1.7α. F0103262CO2, F0103275B05 and F0103387G05 are more subtly affected in terms of EC50 or Bmax by some of the polymorphisms. None of the individual DI S5-S6 polymorphisms by themselves appear to have an impact on the binding of the two rhNav1.7α cross-reactive ISVDs F0103387G04 and F0103464B09. In addition, no effect on binding of the two ISVDs was observed (data not shown) for the three extracellular DIV VSD huNav1.7α-rhNav1.7α polymorphisms Q1530P, H1531Y and E1534D (FIG. 22G).

TABLE 23

CHO
CHO
CHO
CHO

CHO
FlpIn
FlpIn
FlpIn
FlpIn
CHO

FlpIn
huNav1.7α
huNav1.7α
huNav1.7α
huNav1.7α
FlpIn

huNav1.7α +
(F276V) +
(R277Q) +
(E281V) +
(V331M) +
rhNav1.7α +

β1-β2-
β1-β2-β3
β1-β2-β3
β1-β2-β3
β1-β2-
β1-β2-

β3 (SEQ
(SEQ ID
(SEQ ID
(SEQ ID
β3 (SEQ
β3 (SEQ

ID NO: 3)
NO: 5)
NO: 6)
NO: 7)
ID NO: 8)
ID NO: 4)

ID #
EC50 [M]
EC50 [M]
EC50 [M]
EC50 [M]
EC50 [M]
EC50 [M]

F0103265B04
9.2E−09
—
7.1E−09
7.5E−09
—
—

F0103362B08
4.3E−08
4.2E−08
3.4E−08
3.4E−08
—
—

F0103262C02
2.9E−07
2.1E−07
4.5E−07
2.4E−07
8.6E−08
—

F0103262B06
4.7E−08
—
—
—
6.3E−08
—

F010301080*
3.4E−09
2.6E−09
3.2E−09
2.5E−09
—
—

F0103275B05
3.0E−08
2.6E−08
2.5E−08
2.4E−08
2.1E−08
—

F0103387G04
1.1E−08
7.4E−09
7.1E−09
1.1E−08
4.9E−09
5.9E−07

F0103387G05
2.6E−09
2.4E−09
2.2E−09
2.3E−09
5.9E−09
—

F0103464B09
3.4E−09
2.7E−09
2.6E−09
2.7E−09
1.6E−09
3.1E−08

—, no binding;

*F0103265A11(S53W, T57V, N58T, V60N) affinity maturation variant

Summary Epitope Mapping

The combined data of the binding studies on the huNav157 chimeras and the huNav1.7α-rhNav1.7α chimeras, together with the competition binding data suggests that the ISVDs recognize an overlapping epitope on the DI S5-S6 part of huNav1.7α, which can be further delineated by the extracellular human-rhesus polymorphisms in that part which can be further dissected out by the extracellular huNav1.7α-rhNav1.7α polymorphisms in that area or by additional contacts with the adjoining DIV VSD in the case of F0103275B05.

Example 7

Electrophysiological characterization of Nav1.7α selective ISVDs on IonFlux 16 automated patch clamp system (Fluxion Biosciences, Inc., Alameda, CA).

Solutions and ISVDs Handling

The extracellular solution contained (in mM): 138 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, 5.6 glucose (pH 7.2 with NaOH, and 285-290 mOsmolar). Intracellular solution contained (in mM): 5 NaCl, 100 CsF, 45 CsCl, 10 HEPES, 5 EGTA (pH 7.45 with CsOH, and 300-315 mOsmolar). These solutions were freshly made, filtered and stocked for no longer than 6 months at 4° C.

Cell Preparation

HEK Flp-In and CHO Flp-In cells stably expressing the human Nav1.7α channel were generated. Cells were cultured in T-175 cell culture flasks (Greinerbio-one, catalog #660160) using standard cell culture conditions. CHO Flp-In culture medium consists of F12 nutrient mix (Gibco, catalog #31765) containing 10% FBS (Sigma-Aldrich, catalog #F7524), 0.8 mg/mL hygromycin B (Invitrogen, catalog #10687010). HEK Flp-In culture medium consists of DMEM Glutamax™ (Gibco, catalog #31966) containing 10% FBS (Sigma-Aldrich, catalog #F7524), 0.8 mg/mL hygromycin B (Invitrogen, catalog #10687010), 1% NEAA (Gibco, catalog #11140) and 1% Na-pyruvate (Gibco, catalog #11360). Cells were seeded at a density of 1.7×10 4 cells/cm 2 (Hek293 Flp-In) or 5.7×10 3 cells/cm 2 (CHO Flp-In) for 2 days before being used in the IonFlux 16 (Fluxion). Optimal cell confluence prior to harvesting never exceeded 80%. The cells were washed twice with d-PBS without Ca²⁺ and Mg²⁺(Gibco, catalog #14190) and detached with 4 mL Trypsin/EDTA 0.25% (Invitrogen, catalog #25200-056) for 5 to 10 min at 37° C. Medium containing 10% FBS is added to inactivate the enzymatic reaction triggered by the trypsin. Subsequently, the cells were counted (Casy TT, Roche) and centrifuged at 200×g during 2 min at RT in 50 mL conical CELLSTAR® tube (Greiner Bio-One, catalog #227-261) suspended at 1×10 6 cells/ml in CHO—S-SFMII (Gibco, catalog #12052) supplemented with 25 mM Hepes (Gibco, catalog #15630), transferred to a 25 mL cell culture flask (Greiner Bio-One, catalog #690190) and gently shaken at RT for approximately 20 min. 1×10 7 cells were centrifuged for 2 min at 200×g. The pellet is gently resuspended in 5 mL extracellular buffer and centrifuged a second time for 2 min at 200×g. Finally, the pellet is resuspended in 2000 μl extracellular buffer and immediately tested on the IonFlux.

IonFlux Automated Patch Clamp Procedure

250 μL of sterile cell culture grade water is dispensed into every well of the IonFlux 96-well plate except the outlet wells, using an eight channel multi-pipette. Any excess water on the rim of the plate is wiped off before rinsing the plate. The designated plate is inserted into the IonFlux system and subsequently rinsed 4 times according to a standard Water Rinse protocol. After rinsing, the plate is emptied. The inlet wells were then manually filled with extracellular buffer, trap wells with intracellular buffer and the diluted ISVDs or selective peptides were distributed into the compounds wells (250 μL/well). Subsequently, the plate is primed before the actual experiment according to the plate specific protocols. For population plates (Molecular Devices, catalog #910-0098): 1) traps and compounds at 5 psi for t=0-160 s and 2 psi for t=160-175 s, 2) traps but not compounds at two psi for t=175-180 s, and 3) main channel at 1 psi for t=0-160 s and 0.3 psi for t=162-180 s. For single cell plates (Molecular Devices, #910-0100): 1) traps but not compounds at eleven psi for t=0-350 s and 1.5 psi for t=625-630 2) traps and compounds at five psi for t=350-600 s and 1.5 psi for t=600-625 s, and 3) main channel at 0.5 psi for t=0-350 s and one psi for t=350-600 s, and 0.3 psi for t=600-627 s. After priming, the outlet and inlet wells were emptied and 250 μL of the prepared cell suspension (i.e. approximately one million cells) is distributed into the inlet wells of the designated plate. After introduction of the cells, the plate is reprimed: 1) traps and compounds at five psi for t=0-20 seconds and two psi for t=25-50 seconds, 2) traps not with compounds at two psi for t=50-55 seconds, and 3) main channel at one for t=0-30 seconds and 0.4 psi for t=30-55 seconds. Then, cells were introduced to the main channel and trapped at lateral trapping sites with the trapping protocol: 1) trapping vacuum of 7 mmHg for t=0 to 85 seconds, 2) main channel pressure of 0.2 psi for t=0-2 seconds, followed by 15 repeated square pulses of 0-0.2 psi with baseline duration of 4.2 seconds and pulse duration of 0.8 seconds, followed by 0.2 psi for 8 seconds. Whole cell access is achieved by rupturing the patch of the membrane over the hole using the break protocol. A different protocol is used for CHO or HEK293 cells. Breaking protocol for HEK293 cells: 1) breaking vacuum of seven mmHg for t=0-5 seconds, followed by a square pulse of 18 mmHg with a pulse duration of 15 seconds, and followed by 6 mmHg for five seconds, and 2) main channel pressure at 0.15 psi for t=0-25 seconds. Breaking protocol for CHO cells: 1) breaking vacuum of seven mmHg for t=0-10 seconds, followed by a square pulse of 25 mmHg with a pulse duration of five seconds, followed by 6 mmHg for 6 seconds, and a second pulse of 25 mmHg with a pulse duration of five seconds, followed by 6 mmHg for 80 seconds, and 2) main channel pressure at 0.15 psi for t=0-120 seconds. After whole cell configuration, the vacuum pressure is held at 5 mmHg and the main channel pressure at 0.1 psi until the end of the experiment. Cells were first allowed to dialyze for 300 seconds, before compounds were tested. A time course protocol is applied to assess the effect of the compounds on sodium currents elicited by a depolarizing pulse protocol. In order to be able to perform an off-line linear leak subtraction, cells were clamped at −100 mV for 50 milliseconds then hyperpolarized to −120 mV for 100 milliseconds, and repolarized to −80 mV for 30 milliseconds.

Two data acquisition protocols were used: single pulse and two pulse. Single pulse protocol: cells were clamped at a holding potential of −100 mV, stepped to −120 mV for 100 milliseconds to maximize channel availability and then to −30 mV for 50 milliseconds to open the Nat channels. The sweep interval was five seconds with a holding potential of −80 Mv (FIG. 23A). For the two pulse protocol sodium currents were elicited by a depolarizing step from −80 mV to −30 mV for 1000 millieseconds, followed by 10 ms hyperpolarization at −120 mV and a second depolarizing step at −30 mV for 10 milliseconds. The sweep interval was 9 seconds with a holding potential of −80 mV (FIG. 23B).

After the stabilizing period, extracellular buffer is continuously perfused for 120 seconds as a negative control, followed by sequential perfusion of different concentrations of ISVDs or selective peptides. The inhibitory responses were recorded at room temperature (21° C.-24° C.) with a minimum of n=2 for each compound.

IonFlux Data Inclusion Criteria and Data Analysis

Data points were accepted when:

(A) Automated Population Patch

- a. Individual membrane resistance quality and stability is greater than 3 MS2 during data acquisition
- b. Current amplitude quality and stability is greater than 2 nA at −30 mV after negative control
- c. Run-up/run-down less than 10% during data acquisition
- d. IC₅₀value for reference compounds within anticipated range
  
  (B) Automated Single cell patch
- a. Individual membrane resistance quality and stability is greater than 500 MΩ during data acquisition
- b. Current amplitude quality and stability is greater than 200 pA at −30 mV after negative control
- c. Run-up/run-down less than 10% during data acquisition
- d. IC50 value for reference compounds within anticipated range

Currents were measured using IonFlux software (Fluxion Biosciences) and monitored continuously during the exposure to the compounds. Measured currents were normalized by the mean I sustained corrected amplitude prior to compound addition. Current inhibition is estimated by the residual response after 120 seconds of each compound application. Data analysis was performed with IonFlux software (Fluxion Biosciences), Microsoft Excel (Microsoft) and Prism 6 (GraphPad Software).

Electrophysiology Experiments

A series of experiments was performed, using the two pulse protocol shown in FIG. 23B and a single concentration (1 μM) of F0103265B04, F0103265A11, F0103275B05, F0103362B08, F0103387G04, F0103387G05, F0103262CO2, and F0103262B06 applied for 5 minutes to CHO Flpin huNav1.7α+Navβ1-βNav2-Navβ3 cells, HEK293 huNav1.7α+Navβ1 cells, HEK293 huNav1.7α cells and HEK FlpIn huNav1.7α+Navβ1-Navβ2-Navβ3 cells (see FIGS. 25A-25E). In another experiment, a concentration range (1 μM to 1 nM) of F0103265B04 and F0103362B08 was applied to HEK Flpin huNav1.7α+Navβ1-Navβ2-Navβ3 cells, using the same protocol (see FIG. 24). From these experiments it could be concluded that F0103265B04, F0103265A11, F0103275B05, F0103362B08, F0103387G04 and F0103387G05 but not F0103262CO2 or F0103262B06, functionally inhibit huNav1.7α currents in a dose-dependent manner.

After the application of F0103265B04 to the cells was stopped and the compound was allowed to wash out by application of buffer, the cells were continued to be monitored on the patch clamp. The inhibitory effect of F0103265B04 did not wash out in the time frame (11 minutes) of the experiment (see FIG. 26).

A time course experiment with F0103265B04 using the single pulse protocol (see FIG. 25A) revealed that it takes greater than two minutes at 1 μM and greater than eight minutes for F0103265B04 at 10 nM and 100 nM to fully block of the huNav1.7α currents (see FIG. 27).

Example 8

Sequence optimization is a process in which parental ISVD sequences are mutated to yield ISVD sequences that are more identical to human and/or llama/alpaca IGHV3-IGHJ germline consensus sequences. Specific amino acids, with the exception of the so-called hallmark residues, in the FRs that differ between the ISVD and the human IGHV3-IGHJ germline consensus are altered to the human counterpart in such a way that the protein structure, activity and stability are kept intact. In addition, the amino acids present in the CDRs for which there is experimental evidence that they are sensitive to post-translational modifications (PTMs) are altered in such a way that the PTM site is inactivated while the protein structure, activity and stability are kept intact. Furthermore, in order to reduce the binding of pre-existing antibodies to the ISVDs, certain FR residues are altered.

Amino acid residue differences in the CDR regions are not taken into account for sequence optimization. All amino acid differences in the FRs between the ISVD and the human VH341-1 consensus counterparts are identified. Typically, these amino acid residues (numbered according to Kabat) fall into three classes:

1. Hallmarks: These residues are known to be critical for the stability/activity/affinity of the ISVD (based on literature). Therefore, these positions are usually not included in the process. Only when a hallmark is deviating from its llama germline, it is taken into account to be mutated back to the llama/alpaca germline sequence to evaluate potential improvements in stability/activity/affinity. When taken into account this mutation is investigated on an individual basis.

2. Standard: Sequence optimization of these positions is not expected to dramatically change the stability/activity/affinity of the ISVD (based on previous sequence optimization efforts) and they are therefore altered all at once, yielding a basic variant.

3. Unique: It is not known if sequence optimization of these positions affects the stability/activity/affinity of the ISVD and therefore they are investigated on an individual basis on top of the basic variant. These positions typically differ from ISVD to ISVD.

A potential PTM site will only be mutated when there is evidence that the particular site is sensitive to modification under accelerated stress conditions. If a particular amino acid position is insensitive, the parental sequence will be left unchanged in the final construct. Assessment of chemical stability by means of accelerated stress studies is performed by CMC. The N-terminal Glu residue of the first block of an ISVD construct will always be mutated to an Asp (E1D) because experimental evidence has shown that the majority of ISVDs is significantly sensitive to pyroglutamate formation and that the E1D mutation has no effect on stability/activity/affinity of the ISVD. The E1 residues of all other building blocks in the construct are not mutated.

In order to reduce the binding of pre-existing antibodies to the ISVDs, L11V and V89L substitutions are introduced to the FRs and an Ala residue is added to the very C-terminus of the ISVD construct. Exceptionally, the T110K mutation may be introduced as well. The “humanness” of a sequence optimized ISVD may be defined as:

Percent amino acid identity in the FRs of the ISVD vs the human VH3-JH consensus sequence

wherein the CDRs may be defined by Kabat, IMGT, AbM, Chothia, or the like. In particular embodiments, the calculation is performed in which the CDRs are defined by at least two methods.

Sequence Optimization of F0103275B05/F0103387G04

Several PTM substitution libraries were generated based on the accelerated stress data summarized in Table 24 and screened as crude periplasmic extracts in binding FACS on human and rhesus Nav1.7α. FIG. 28 shows a sequence analysis of F0103275B05/387G04 aligned against the human VH3-J3 consensus sequence and the llama VHH2 consensus sequence.

TABLE 24

Results accelerated stress experiments performed on F0103275B05/387G04 variants

Stress
Modification

ID #
Description
Site
condition
observed

F010300659
F0103275B05(S27P, I28V,
NA
1 week @, 45°
0.2% increase

S50Y, N53P, G55W, S56D,

C., ±1 mg/mL
of pre-peak

T57W, N93R, A94W) +

in D-PBS
(SE-HPLC)

FLAG3-HIS6

F010301452
F0103275B05(S27P, I28V,
NA
1 week @ 45°
0.1% increase

S50Y, N53P, G55W, S56D,

C., ±1 mg/mL
of pre-peak

T57W, N93R, A94W)

in D-PBS
(SE-HPLC)

F010301457
F0103275B05(S27P, I28V,
N32
4 weeks @ −20,
Not sensitive

S50Y, N53P, G55W, S56D,

25 and 40° C.

T57W, N93R, A94W) + HIS6

F010301457
F0103275B05(S27P, I28V,
N73
4 weeks @, −20,
57%

S50Y, N53P, G55W, S56D,

25 and 40° C.

T57W, N93R, A94W) + HIS6

F010301894
F0103387G04(L11V, A12V,
D72
4 weeks @ −20,
1.8%-12.3%

K33R, R39Q, S50Y, S56D,

25 and 40° C.

T60A, R76N, W78V, S79Y,

T83R, V89L, N93R) + HIS6

F010301894
F0103387G04(L11V, A12V,
N100c
4 weeks @ −20,
1.4-8.9%

K33R, R39Q, S50Y, S56D,

25 and 40° C.

T60A, R76N, W78V, S79Y,

T83R, V89L, N93R) + HIS6

F010301895
F0103387G04 + HIS6
D72
4 weeks @ −20,
0.1%-1.7%

25 and 40° C.

F010301895
F0103387G04 + HIS6
N100c
4 weeks @ −20,
0.7-2.7%

25 and 40° C.

F010301950
F0103387G04(L11V, A12V,
M34
10 mM H₂O₂
5%

K33R, R39Q, S50Y, S56D,

for 3 h @ RT

T60A, W78V, S79Y, T83R,

V89L, N93R) + HIS6

F010301950
F0103387G04(L11V, A12V,
D72
4 weeks @ −20,
3-15%

K33R, R39Q, S50Y, S56D,

25 and 40° C.

T60A, W78V, S79Y, T83R,

V89L, N93R) + HIS6

F010301950
F0103387G04(L11V, A12V,
N100c
4 weeks @ −20,
1.2-8.1%

K33R, R39Q, S50Y, S56D,

25 and 40° C.

T60A, W78V, S79Y, T83R,

V89L, N93R) + HIS6

F010301950
F0103387G04(L11V, A12V,
D99
4 weeks @ −20,
0.5-4.3%

K33R, R39Q, S50Y, S56D,

25 and 40° C.

T60A, W78V, S79Y, T83R,

V89L, N93R) + HIS6

F010302383
F0103387G04(L11V, A12V,
NA
1 week @ 45°
0.2% increase

K33R, R39Q, S50Y, S56D,

C., ±1 mg/mL
of pre-peak

T60A, D72G, W78V, S79Y,

in D-PBS
(SE- HPLC)

T83R, V89L, N93R, D99S,

N100cG) + FLAG3-HIS6

NA, not applicable

Screening of F0103275B05/387G04 PTM Substitution Libraries

Several PTM substitution libraries were generated based on the accelerated stress data summarized in Table 24 and screened as crude periplasmic extracts in binding FACS on human and rhesus Nav1.7α.

- N73G substitution resulted in a comparable or slightly improved binding profile compared to the parental reference F0103275B05 (Table 25) and was retained as this was also the naturally occurring residue on this position in F0103387G04 (FIG. 28).
- Both D72G and D72Q substitutions resulted in a comparable or slightly improved binding profile compared to the parental reference (Table 27) and were further evaluated, as well G73A and G73R substitutions.
- D99S, D99R and D99N substitutions resulted in a comparable or slightly improved binding profile compared to the parental reference (Table 26) and were further evaluated, as well as S100R and S100V substitutions; S99 is the naturally occurring residue on this position in F0103275B05 (FIG. 28).
- N100cI and N100cG (Kabat position 100c) substitutions resulted in a comparable or slightly improved binding profile compared to the parental reference (Table 27) and were further evaluated; I100c is the naturally occurring residue on this position in F0103275B05 (FIG. 28)

TABLE 25

Summary screening N73X & A74X substitution libraries

CHO Flp-In
CHO Flp-In

huNav1.7α + β1-β2-β3
rhNav1.7α + β1-β2-β3

(SEQ ID NO: 3)
(SEQ ID NO: 4)

Mean
SD
Mean
SD

Substitution
MFI
MFI
MFI
MFI

A74C
1371
178
1380
148

A74D
8876
623
10933
506

A74E
6156
485
6767
677

A74F
11022
799
16436
605

A74G
12165
1360
18665
1369

A74H
12477
2828
18686
4367

A74I
4945
956
7560
1518

A74K
15407
1520
18490
1754

A74L
7173
1605
10190
1736

A74N
18129
463
26491
518

A74P
8928
2193
14486
3731

A74Q
9585
912
14644
1030

A74R
11403
43
16344
1503

A74S
10010
1672
15420
3795

A74T
9496
70
13521
666

A74V
6136
3294
9381
4939

A74W
9065
680
14913
922

A74Y
14759
324
21008
808

Blanc
515
37
482
35

Parental reference
10260
2842
15695
4311

Negative control
516
47
560
149

N73A
9387
2705
14029
3929

N73C
1396
8
1910
66

N73E
6536
478
9752
768

N73F
6341
714
9340
1556

N73G
18297
4375
27008
4900

N73H
10773
105
16937
830

N73I
4274
1138
6184
1751

N73K
13248
1558
17919
1854

N73L
4640
306
6839
199

N73M
5088
7
7638
112

N73P
4910
345
6851
610

N73Q
6614
1334
9716
2509

N73R
11583
1642
17083
2197

N73S
12569
2453
19307
3068

N73T
8839
279
14387
99

N73V
4827
185
7467
91

N73W
5829
882
8444
1061

N73Y
5732
1146
8745
1594

TABLE 26

Summary screening D99X & S100X substitution libraries

CHO Flp-In
CHO Flp-In

huNav1.7α + β1-β2-β3
rhNav1.7α + β1-β2-β3

(SEQ ID NO: 3)
(SEQ ID NO: 4)

Mean
SD
Mean
SD

Substitution
MFI
MFI
MFI
MFI

D99A
14187
4136
9871
2193

D99C
4625
803
1637
418

D99E
10633
2657
6153
1878

D99F
23749
3820
1834
255

D99G
23305
4036
10611
1456

D99H
23969
8888
11169
3971

D99I
4646
726
551
9

D99K
46514
619
1036
31

D99L
12797
251
742
103

D99M
15591
1834
1136
98

D99N
41774
971
45539
49

D99P
568
6
518
10

D99R
45426
7088
9917
2228

D99S
27582
442
32383
2312

D99V
12103
3467
7536
1541

D99W
23646
5065
8717
2202

D99Y
36537
6107
11155
2438

Parental reference
21107
6057
24705
5475

S100A
17866
3265
18065
3864

S100C
10920
2106
10646
3547

S100D
3674
572
2003
206

S100E
9252
711
6475
1144

S100F
30229
410
27675
1566

S100G
22114
5108
22287
6403

S100K
48169
1478
3966
153

S100L
18824
2146
21105
437

S100M
26478
1935
30669
2530

S100Q
33974
2862
29579
1025

S100R
46564
11444
12496
2135

S100T
23257
7442
23861
5720

S100V
29079
3063
30108
330

S100W
16328
1359
11736
1568

S100Y
29104
1100
18385
1265

TABLE 27

Summary screening D72X, N100cX

& T100dX substitution libraries

CHO Flp-In
CHO Flp-In

huNav1.7α + β1-β2-β3
rhNav1.7α + β1-β2-β3

(SEQ ID NO: 3)
(SEQ ID NO: 4)

Mean
SD
Mean
SD

Substitution
MFI
MFI
MFI
MFI

Parental reference
20313
8867
21158
8172

Blanc
516
21
462
19

D72A
32097
33
32753
1387

D72C
17787
10134
18066
8851

D72E
30260
14879
29532
13937

D72F
44931
24269
41549
17306

D72G
45410
7433
40171
4018

D72I
49815
15573
45508
12208

D72K
38860
3520
37695
3863

D72L
25424
11275
25338
9201

D72M
31103
13565
30170
11470

D72P
33230
11366
33325
8976

D72Q
47752
4735
43479
6177

D72T
38112
13534
35858
10233

D72V
31046
1671
30086
2155

D72W
44290
19513
38836
16236

D72Y
48641
19181
44618
14962

N100cA
18336
4353
16832
3604

N100cD
33726
3075
25954
3742

N100cE
21563
4451
15559
4511

N100cF
49642
6758
45764
6911

N100cG
54396
3864
46627
6061

N100cI
48332
2499
45805
2003

N100cK
46602
9571
50105
7223

N100cL
22679
3863
21605
3558

N100cM
36747
5344
35809
3787

N100cP
42552
379
35496
130

N100cQ
31049
966
28498
371

N100cR
39701
8211
46177
4971

N100cS
45457
167
43443
390

N100cT
38337
13505
35636
12150

N100cV
23442
3541
23509
3556

N100cW
38400
11435
37044
6489

N100cY
26791
303
27111
348

T100dA
8815
4912
5014
2542

T100dC
7049
1662
8567
2118

T100dD
3647
1652
6727
3269

T100dE
541
12
1562
384

T100dF
628
133
8704
7843

T100dH
19494
9467
15424
7021

T100dI
884
161
24959
17119

T100dK
519
39
462
31

T100dL
807
34
19047
1558

T100dM
7096
10045
35198
14877

T100dP
2934
840
20174
6801

T100dQ
700
75
7062
3009

T100dR
529
6
464
10

T100dS
20718
6040
23029
5954

T100dV
5499
611
13598
1603

T100dW
638
4
5362
810

T100dY
937
166
26415
9982

Characterization of F0103275B05/387G04 Variants

Sequence optimization was initiated on F0103275B05 (Table 29) but later on continued on the related and improved F0103387G04 (Table 30). Likewise, affinity maturation substitutions identified for F0103275B05 were successfully transferred to F0103387G04. The variants were compared in binding FACS on human and rhesus Nav1.7α, in aSEC for possible multimerization, in OD340 for insoluble aggregate formation and in the thermal shift assay for Tm.

The thermal shift assay (TSA) was performed in a 96-well plate on the LightCycler 48011 machine (Roche). Per row, one sample was analyzed according to the following pH range: 3.5/4/4.5/5/5.5/6/6.5/7/7.5/8/8.5/9. Per well, 5 μl of sample (0.8 mg/ml in PBS) was added to 5 μL of Sypro Orange (40× in MilliQ water; Invitrogen cat. No. 56551) and 10 μL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate and 115 mM NaCl with a pH ranging 3.5 to 9). The applied temperature gradient (37 to 99° C. at a rate of 0.03° C./s) induces unfolding of the ISVDs whereby their hydrophobic patches become exposed. Sypro Orange binds to those hydrophobic patches, resulting in an increase in fluorescence intensity (Ex/Em=465/580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 serves as a measure of the melting temperature (Tm).

Table 28 summarizes the effects of the explored substitutions.

TABLE 28

Overview of F0103387G04 substitutions

huNav1.7α
rhNav1.7α

EC50
EC50
Tm
aSEC

fold change
fold change
difference
behavior
OD340

compared to
compared to
compared to
compared to
compared to
Retain

Substitution
reference
reference
reference
reference
reference
substitution

L11V
=
=
−2
=
=
Y

(L11V, T83R, V89L)

A12V
ND
ND
ND
ND
ND
Y

K33R
=
+
−4
=
=
Y

(K33R, S50Y, S56D,

N93R)

R39Q
−
−
+3
=
=
Y

S50Y
=
+
−4
=
=
Y

(K33R, S50Y, S56D,

N93R)

S56D
=
+
−4
=
=
Y

(K33R, S50Y, S56D,

N93R)

T60A
=
=
+3
=
=
Y

D72G
+
+
ND
ND
ND
Y

D72Q
+
+
ND
ND
ND
N

G73N
−2
−2
−2
=
=
N

G73A
−
−
−3
=
=
N

G73R
+
+
−4
=
=
N

R76N
−2
−2
−2
=
=
N

R76_V78insT
−3
−6
+5 in
=
=
N

275B05

−5 in

387G04

W78V
=
=
−6
=
=
Y

S79Y
=
=
=
=
=
Y

T83R
=
=
−2
=

Y

(L11V, T83R, V89L)

V89L
=
=
−2
=
=
Y

(L11V, T83R, V89L)

N93R
=
++
−4
=
=
Y

(K33R, S50Y, S56D,

N93R)

D99S
+2
+2
−1
=
=
Y

D99R
+
+
proteolytic degradation
N

D99N
+2
+2
−2
=
=
N

S100R
+
+
proteolytic degradation
N

S100V
−2
−2
+1
=
=
N

N100cG
+
+
=
=
=
Y

N100cI
−
−
−1
=
=
N

ND, not determined

TABLE 29

Characterization of F0103275B05 variants

Part 1

CHO

FlpIn
CHO

huNav1.7α-
FlpIn

β1-β2-
rhNav1.7α-

(SEQ ID
β1-β2-β3

NO: 3)
(SEQ ID

β3
NO: 4)

ID #
L11
S33R
R39
S50Y
S56D
R76
77
T83
V89
N93R
EC50 [M]
EC50 [M]

F010301461
.
R
.
Y
D
.
—
.
.
R
ND
ND

F010301635
V
.
Q
.
.
.
—
R
L
.
ND
ND

F010301636
V
.
.
.
.
N
—
R
L
.
ND
ND

F010301637
V
.
.
.
.
.
—
R
L
.
5.6E−08
ND

F010301638
V
.
Q
.
.
N
—
R
L
.
ND
ND

F010301639
V
.
Q
.
.
.
T
R
L
.
ND
ND

F010301640
V
.
.
.
.
.
T
R
L
.
ND
ND

F010301641
V
.
.
.
.
N
T
R
L
.
ND
ND

F010301642
V
.
Q
.
.
N
T
R
L
.
ND
ND

F010301652
V
R
.
Y
D
N
—
R
L
R
6.1E−08
2.4E−08

F010301653
V
R
.
Y
D
.
—
R
L
R
3.6E−08
1.6E−08

F010301654
V
R
Q
Y
D
.
—
R
L
R
3.2E−08
1.8E−08

F010301655
V
R
Q
Y
D
N
—
R
L
R
4.6E−08
2.6E−08

F0103275B05
.
.
.
.
.
.
—
.
.
.
6.6E−08
—

Part 2

HEKa/b 1

HEK FlpIn
HEK
(SEQ ID

Nav157ch14-
FlpIn
NO: 40)
HEKa

β1-β2-β3
Nav157ch14
huNav1.7α
huNav1.7α

(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID

NO: 20)
NO: 20)
NO: 1)
NO: 1)
Tm

ID #
EC50 [M]
EC50 [M]
EC50 [M]
EC50 [M]
[° C.]

F010301461
ND
ND
ND
ND
64

F010301635
3.5E−08
1.6E−08
3.1E−08
1.9E−08
68

F010301636
4.5E−08
1.4E−08
3.0E−08
1.7E−08
64

F010301637
2.9E−08
1.1E−08
3.0E−08
1.3E−08
66

F010301638
5.0E−08
1.8E−08
3.2E−08
2.3E−08
67

F010301639
—
—
9.4E−08
—
72

F010301640
—
—
6.5E−08
—
69

F010301641
3.5E−07
3.1E−07
2.3E−07
3.8E−07
70

F010301642
—
—
—
—
73

F010301652
ND
ND
3.8E−08
2.4E−08
60

F010301653
ND
ND
2.9E−08
1.5E−08
62

F010301654
ND
ND
2.5E−08
1.8E−08
65

F010301655
ND
ND
2.8E−08
1.9E−08
63

F0103275B05
3.3E−08
1.2E−08
3.9E−08
1.4E−08
68

ND, not determined

TABLE 30

Characterization of F0103387G04 variants

Part 1

ID #
L11
A12
K33
R39
S50
S56
T60
D72
G73
R76

F010301656
.
.
R
.
Y
D
.
.
.
.

F010301840
V
V
R
Q
Y
D
.
.
.
.

F010301841
V
V
R
Q
Y
D
.
.
.
.

F010301842
V
V
R
Q
Y
D
A
.
.
.

F010301843
V
V
R
Q
Y
D
.
.
N
.

F010301844
V
V
R
Q
Y
D
.
.
.
N

F010301845
V
V
R
Q
Y
D
.
.
.
.

F010301846
V
V
R
Q
Y
D
.
.
.
.

F010301847
V
V
R
Q
Y
D
A
.
N
N

F010301848
V
V
R
Q
Y
D
A
.
N
.

F010301865
V
V
R
Q
Y
D
A
.
.
.

F010301866
V
V
R
Q
Y
D
A
.
.
N

F010302310
V
V
R
Q
Y
D
A

A
.

F010302311
V
V
R
Q
Y
D
A

R
.

F010302312
V
V
R
Q
Y
D
A
.
.
.

F010302313
V
V
R
Q
Y
D
A
.
A
.

F010302314
V
V
R
Q
Y
D
A
.
R
.

F010302315
V
V
R
Q
Y
D
A

.
.

F010302316
V
V
R
Q
Y
D
A

A
.

F010302317
V
V
R
Q
Y
D
A

R
.

F010302318
V
V
R
Q
Y
D
A

.
.

F010302319
V
V
R
Q
Y
D
A

A
.

F010302320
V
V
R
Q
Y
D
A
.
A
.

F010302321
V
V
R
Q
Y
D
A

R
.

F010302322
V
V
R
Q
Y
D
A

.
.

F010302323
V
V
R
Q
Y
D
A

A
.

F010302324
V
V
R
Q
Y
D
A

R
.

F010302325
V
V
R
Q
Y
D
A
.
R
.

F010302326
V
V
R
Q
Y
D
A

.
.

F010302327
V
V
R
Q
Y
D
A

A
.

F010302328
V
V
R
Q
Y
D
A

R
.

F010302329
V
V
R
Q
Y
D
A

.
.

F010302330
V
V
R
Q
Y
D
A

A
.

F010302331
V
V
R
Q
Y
D
A

R
.

F010302332
V
V
R
Q
Y
D
A

.
.

F010302370
V
V
R
Q
Y
D
A
.
R
.

F010302371
V
V
R
Q
Y
D
A
.
R
.

F010302372
V
V
R
Q
Y
D
A
.
R
.

F010302383
V
V
R
Q
Y
D
A
G
.
.

F010302384
V
V
R
Q
Y
D
A
G
.
.

F010302385
V
V
R
Q
Y
D
A
Q
.
.

F010302386
V
V
R
Q
Y
D
A
Q
.
.

F0103387G04
.
.
.
.
.
.
.
.
.
.

Part 1

ID #
77
W78
S79
T83
V89
N93
D99
S100
N100c

F010301656
—
.
.
.
.
R
.
.
.

F010301840
T
.
.
R
L
R
.
.
.

F010301841
—
.
.
R
L
R
.
.
.

F010301842
—
.
.
R
L
R
.
.
.

F010301843
—
.
.
R
L
R
.
.
.

F010301844
—
.
.
R
L
R
.
.
.

F010301845
—
V
.
R
L
R
.
.
.

F010301846
—
.
Y
R
L
R
.
.
.

F010301847
—
V
Y
R
L
R
.
.
.

F010301848
—
V
Y
R
L
R
.
.
.

F010301865
—
V
Y
R
L
R
.
.
.

F010301866
—
V
Y
R
L
R
.
.
.

F010302310
—
V
Y
R
L
R
R
R
.

F010302311
—
V
Y
R
L
R
R
R
.

F010302312
—
V
Y
R
L
R
.
.
I

F010302313
—
V
Y
R
L
R
.
.
I

F010302314
—
V
Y
R
L
R
.
.
I

F010302315
—
V
Y
R
L
R
R
.
I

F010302316
—
V
Y
R
L
R
R
.
I

F010302317
—
V
Y
R
L
R
R
.
I

F010302318
—
V
Y
R
L
R
.
R
I

F010302319
—
V
Y
R
L
R
.
R
I

F010302320
—
V
Y
R
L
R
.
.
.

F010302321
—
V
Y
R
L
R
.
R
I

F010302322
—
V
Y
R
L
R
R
R
I

F010302323
—
V
Y
R
L
R
R
R
I

F010302324
—
V
Y
R
L
R
R
R
I

F010302325
—
V
Y
R
L
R
.
.
.

F010302326
—
V
Y
R
L
R
R
.
.

F010302327
—
V
Y
R
L
R
R
.
.

F010302328
—
V
Y
R
L
R
R
.
.

F010302329
—
V
Y
R
L
R
.
R
.

F010302330
—
V
Y
R
L
R
.
R
.

F010302331
—
V
Y
R
L
R
.
R
.

F010302332
—
V
Y
R
L
R
R
R
.

F010302370
—
V
Y
R
L
R
S
.
I

F010302371
—
V
Y
R
L
R
N
V
I

F010302372
—
V
Y
R
L
R
.
V
I

F010302383
—
V
Y
R
L
R
S
.
G

F010302384
—
V
Y
R
L
R
S
.
I

F010302385
—
V
Y
R
L
R
S
.
G

F010302386
—
V
Y
R
L
R
S
.
I

F0103387G04
—
.
.
.
.
.
.
.
.

Part 2

CHO

FlpIn
CHO
CHO
CHO

huNav1.7α +
FlpIn
FlpIn
FlpIn

β1-β2-
huNav1.7α +
rhNav1.7α-
rhNav1.7α-

β3 (SEQ
β1-β2-
β1 + β2-
β1 + β2-

ID NO:
β3(SEQ
β3 (SEQ
β3 (SEQ

3) EC50
ID NO: 3)
ID NO: 4)
ID NO:

ID #
[M]
Bmax
EC50 [M]
4) Bmax
Tm [° C.]

F010301656
1.7E−08
100%
8.0E−09
100%
72

F010301840
4.7E−08
100%
4.8E−08
50%
70

F010301841
1.4E−08
100%
7.6E−09
100%
75

F010301842
1.5E−08
100%
7.9E−09
100%
78

F010301843
1.8E−08
100%
9.3E−09
100%
74

F010301844
2.3E−08
100%
1.1E−08
100%
73

F010301845
1.7E−08
100%
8.0E−09
100%
69

F010301846
2.1E−08
100%
1.1E−08
100%
75

F010301847
6.7E−08
100%
2.9E−08
100%
70

F010301848
4.4E−08
100%
1.9E−08
100%
73

F010301865
2.0E−08
100%
1.1E−08
100%
75

F010301866
3.8E−08
100%
1.8E−08
100%
73

F010302310
ND
ND
ND
ND
ND

F010302311
ND
ND
ND
ND
ND

F010302312
2.4E−08
100%
1.8E−08
100%
74

F010302313
3.8E−08
100%
3.4E−08
96%
71

F010302314
1.6E−08
100%
1.4E−08
98%
70

F010302315
7.2E−09
100%
3.9E−08
23%
ND

F010302316
8.2E−09
91%
—
—
ND

F010302317
6.8E−09
91%
1.9E−08
13%
ND

F010302318
1.1E−08
89%
—
—
ND

F010302319
1.5E−08
88%
—
—
ND

F010302320
3.0E−08
99%
2.1E−08
100%
71

F010302321
7.9E−09
91%
—
—
ND

F010302322
ND
ND
ND
ND
ND

F010302323
ND
ND
ND
ND
ND

F010302324
2.9E−08
42%
—
—
ND

F010302325
1.3E−08
96%
8.7E−09
99%
71

F010302326
6.2E−09
89%
1.8E−08
32%
ND

F010302327
7.0E−09
87%
9.3E−08
7%
ND

F010302328
ND
ND
ND
ND
ND

F010302329
7.6E−09
94%
1.9E−08
44%
ND

F010302330
1.0E−08
93%
2.0E−07
23%
ND

F010302331
6.0E−09
90%
2.4E−08
38%
ND

F010302332
ND
ND
ND
ND
ND

F010302370
9.0E−09
100%
7.3E−09
100%
69

F010302371
9.4E−09
100%
1.0E−08
100%
68

F010302372
1.7E−08
100%
2.0E−08
100%
70

F010302383
6.8E−09
100%
4.5E−09
100%
73

F010302384
6.2E−09
100%
4.3E−09
100%
72

F010302385
7.9E−09
100%
5.0E−09
100%
74

F010302386
7.3E−09
100%
4.9E−09
100%
72

F0103387G04
1.7E−08
100%
8.7E−08
10%
76

ND, not determined

Selection of an F0103387G04 Sequence Optimization Variant

Variant F010302383 was selected as the final sequence optimization variant of F0103387G04 (see F0103387G04 SO in FIG. 28). It boasts a 2- and 20-fold improved binding on huNav1.7α and rhNav1.7α respectively, as well as comparable aSEC and OD340 nm behavior and a slightly reduced thermal stability (Table 31). In vitro electrophysiology experiments confirmed the low nM potency on huNav1.7α and rhNav1.7α and selectivity over Nav1.4, Nav1.5α and Nav1.6α. All PTM liabilities (Table 24) were successfully substituted. Cell-free fermentation expression titers at CMC of the corresponding monovalent tagless variant F01032396 in P. pastoris were 3.6 g/L.

TABLE 31

Sequence optimization variant of F0103387G04

Part 1

CHO
CHO
CHO

FlpIn
FlpIn
FlpIn
HEK
HEK

huNav1.7α +
rhNav1.7α +
rhNav1.7α +
huNav1.7α +
rhNav1.7α +
HEK
HEK
HEK

β1-β2-β3
β1-β2-β3
β1-β2-β3
β1
β1-β2-β3
huNav1.4α
huNav1.5α
huNav1.6α

(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID

NO: 3)
NO: 4)
NO: 4)
NO: 44)
NO: 4)
NO: 1)
NO: 27)
NO: 28)

ID #
EC50 [M]
EC50 [M]
Bmax
IC50 [M]
IC50 [M]
IC50 [M]
IC50 [M]
IC50 [M]

F0103387G04
1.7E−08
8.7E−08
10%
ND
ND
ND
ND
ND

F010302383
6.8E−09
4.5E−09
100%
2.2E−08
3.0E−09
—
—
—

—, no activity observed @ 7 μM;

ND, not determined

Part 2

ID #
Tm [° C.]
Tagg [° C.]
aSEC
OD340 nm
AbM
Kabat

F0103387G04
76
ND
ok
ok
81%
78%

F010302383
73
69
ok
ok
83%
79%

ND, not determined

Example 9
Sequence Optimization of F0103387G05

Several PTM substitution libraries were generated based on the accelerated stress data summarized in Table 32 and screened as crude periplasmic extracts in binding FACS on human Nav1.7α.

TABLE 32

Results accelerated stress experiments performed on F0103387G05 variants

Modification

ID #
Description
Site
Stress condition
observed

F0103387G05
F0103387G05-
NA
1 week @ 45°
0.3% increase

FLAG3-HIS6

C., ±1 mg/mL
of pre-peak

in D-PBS
(SE-HPLC)

F010301456
F0103387G05-HIS6
CD
4 weeks @ −20,
0.8-6.1%

R2
25 and 40° C.
isomerization

F010301456
F0103387G05-HIS6
N73
4 weeks @ −20,
1.2-13%

25 and 40° C.

F010301949
F0103387G05(L11V,
CD
4 weeks @ −20,
1-9%

A14P, D23A, H37Y,
R2
25 and 40° C.
isomerization

G40A, A41P, D58G,

N82bS, N83R, V89L,

R105Q)-HIS6

F010301949
F0103387G05(L11V,
N73
4 weeks @ −20,
0.7-9%

A14P, D23A, H37Y,

25 and 40° C.

G40A, A41P, D58G,

N82bS, N83R,

V89L, R105Q)-

HIS6

F010302391
F0103387G05(L11V,
NA
1 week @ 45°
0.2% increase

A14P, D23A, H37Y,

C., ±1 mg/mL
of pre-peak

G40A, A41P, D53G,

in D-PBS
(SE-HPLC)

D54G, D58G, N82bS,

N83R, V89L, R105Q)-

FLAG3-HIS6

ND, not determined

- N73Q substitution resulted in a better binding profile compared to the parental reference (Table 33) and was further evaluated, as well as N73A and N73Y substitutions.
- Because of the available choices to substitute N73, no mutations for A74 were further evaluated.

TABLE 33

Summary screening of N73X & A74 substitution libraries

CHO Flp-In

huNav1.7α + β1-β2-β3

(SEQ ID NO: 3)

Mean
SD

Substitution
MFI
MFI

A74D
72131
8952

A74E
67339
6350

A74I
76360
7842

A74K
99046
1123

A74L
70981
6848

A74N
80896
11713

A74P
63748
2925

A74Q
75053
9885

A74S
57716
7270

A74T
66295
10425

A74W
40642
8566

Blanc
570
NA

Negative control
566
12

Parental reference
48046
8443

N73A
84208
12907

N73D
77600
8148

N73E
64666
7724

N73F
60283
14931

N73G
84896
8452

N73H
68524
2512

N73I
81380
16558

N73K
85809
21302

N73L
70525
14051

N73M
87197
8041

N73P
58705
45951

N73Q
79497
12894

N73R
66654
8484

N73S
85111
2933

N73T
71638
13462

N73V
84743
7727

N73Y
86218
8813

HEK293 human Nav1.7α

(SEQ ID NO: 1)

Mean
SD

Substitution
MFI
MFI

A74D
33271
2071

A74E
28963
2627

A74I
33074
3560

A74K
38376
722

A74L
27756
4959

A74N
34952
2025

A74P
28080
948

A74Q
31394
3986

A74S
29160
2997

A74T
30375
4492

A74W
20460
4869

Blanc
734
NA

Negative control
723
2

Parental reference
22340
3535

N73A
35560
5590

N73D
32744
2730

N73E
29576
3544

N73F
27251
5503

N73G
35331
3769

N73H
31210
1722

N73I
36497
5563

N73K
32267
6201

N73L
34819
2876

N73M
38463
2348

N73P
25112
18794

N73Q
33609
3748

N73R
30872
4401

N73S
36285
1224

N73T
32687
4647

N73V
37400
1183

N73Y
36734
5290

HEK293 human

Nav1.7α + β1

(SEQ ID NO: 44)

Mean
SD

Substitution
MFI
MFI

A74D
79560
6225

A74E
74537
1165

A74I
76639
1998

A74K
84166
964

A74L
75554
2070

A74N
81639
5349

A74P
66190
2235

A74Q
78895
5333

A74S
65093
7925

A74T
72355
5048

A74W
49009
11489

Blanc
789
NA

Negative control
778
6

Parental reference
59514
8475

N73A
83244
10774

N73D
82260
3026

N73E
76352
3918

N73F
64305
10544

N73G
79190
3307

N73H
70459
3060

N73I
79431
7248

N73K
79446
16278

N73L
75337
11011

N73M
84223
4072

N73P
60089
45450

N73Q
78260
5434

N73R
67882
4153

N73S
79692
2217

N73T
76277
8140

N73V
84341
3090

N73Y
85910
1947

NA, not applicable

Characterization of F0103387G05 Variants

Affinity maturation substitutions that improved the binding of F0103387G05 were transferred to the sequence optimized variants. These variants were compared (Table 35) in binding FACS on human Nav1.7α, in aSEC for possible multimerization, in OD340 for insoluble aggregate formation and in the thermal shift assay for Tm. Table 34 summarizes the effects of the explored substitutions.

TABLE 34

Overview of F0103387G05 substitutions

Part 1

huNav1.7α + β1
huNav1.7α
huNav1.7α

(SEQ ID NO: 44)
(SEQ ID NO: 1)
(SEQ ID NO: 1)

EC50
without EC50
without Bmax

fold change
fold change
fold change

compared to
compared to
compared to

Substitution
reference
reference
reference

L11V(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

A14P(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

D23A
+
+
=

H37Y
=
=
=

G40A
=
=
=

A41P
=
=
=

F47L
=
−2
−

D53G (D53G, D54G)
+
+
=

D54G (D53G, D54G)
+
+
=

D58G
+
+
=

N73A
=
=
=

N73Q
=
=
=

N73Y
=
=
=

N82bS(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

N83R(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

V89L(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

E93N
=
−20
−4

R105Q(L11V, A14P, N82bS,
=
=
=

N83R, V89L, R105Q)

Part 2

Tm
aSEC

difference
behavior
OD340

compared to
compared to
compared to
Retain

Substitution
reference
reference
reference
substitution

L11V(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

A14P(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

D23A
+4
=
=
Y

H37Y
=
=
=
Y

++ at low

pH (FIG. 3)

G40A
+1
=
=
Y

A41P
+1
=
=
Y

F47L
−4
=
=
N

D53G (D53G, D54G)
−4
ND
ND
Y

D54G (D53G, D54G)
−4
ND
ND
Y

D58G
−4
=
=
Y

N73A
=
=
=
N

N73Q
=
=
=
N

N73Y
−1
=
=
N

N82bS(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

N83R(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

V89L(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

E93N
−6
=
=
N

R105Q(L11V, A14P, N82bS,
−2
=
=
Y

N83R, V89L, R105Q)

ND, not determined

TABLE 35

Characterization of F010387G05 variants

Part 1

ID #
L11
A14
D23
H37
G40
A41
F47
D53
D54
D58
N73
N82b
N83
V89
E93
R105

F010301556
.
.
A
.
.
.
.
G
G
G
.
.
.
.
.
.

F010301563
.
.
A
.
.
.
.
.
.
G
.
.
.
.
.
.

F010301643
Q
P
A
.
.
.
.
.
.
.
.
S
R
L
.
Q

F010301644
V
P
.
Y
.
.
.
.
.
.
.
S
R
L
.
Q

F010301645
V
P
.
.
A
.
.
.
.
.
.
S
R
L
.
Q

F010301646
V
P
.
.
.
P
.
.
.
.
.
S
R
L
.
Q

F010301647
V
P
.
.
.
.
L
.
.
.
.
S
R
L
.
Q

F010301648
V
P
.
.
.
.
.
.
.
.
.
S
R
L
N
Q

F010301649
V
P
.
.
.
.
.
.
.
.
.
S
R
L
.
Q

F010301849
V
P
A
.
A
P
.
.
.
G
.
S
R
L
.
Q

F010301850
V
P
A
Y
A
P
.
.
.
G
.
S
R
L
.
Q

F010302307
V
P
A
Y
A
P
.
.
.
G
A
S
R
L
.
Q

F010302308
V
P
A
Y
A
P
.
.
.
G
Y
S
R
L
.
Q

F010302309
V
P
A
Y
A
P
.
.
.
G
Q
S
R
L
.
Q

F010302391
V
P
A
Y
A
P
.
G
G
G
.
S
R
L
.
Q

F010302392
V
P
A
Y
A
P
.
G
G
G
Q
S
R
L
.
Q

F0103387G05
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

Part 2

HEK

HEKa/β1

HEK

FlpIn
HEK

(SEQ ID

FlpIn

Nav157ch14-
FlpIn
HEK
NO: 40)
HEKa

Nav1.7α +

β1-β2-
Nav157ch14
FlpIn
Nav1.7α
Nav1.7α
HEKa
β1- β2-

β3 (SEQ
(SEQ ID
Nav157ch14
(SEQ ID
(SEQ ID
Nav1.7α
β3 (SEQ

ID NO:
NO: 20)
(SEQ ID
NO: 1)
NO: 1)
(SEQ ID
ID NO: 3)

20) EC50
EC50
NO: 19)
EC50
EC50
NO: 1)
EC50

ID #
[M]
[M]
Bmax
[M]
[M]
Bmax
[M]

F010301556
ND
ND
ND
1.4E−09
1.8E−09
ND
ND

F010301563
ND
ND
ND
1.0E−09
1.5E−09
ND
ND

F010301643
3.6E−09
3.7E−09
100%
3.4E−09
4.1E−09
100%
ND

F010301644
4.9E−09
4.4E−09
100%
5.0E−09
5.7E−09
100%
ND

F010301645
4.6E−09
3.9E−09
100%
4.2E−09
4.7E−09
100%
ND

F010301646
4.2E−09
3.1E−09
100%
4.1E−09
4.4E−09
100%
ND

F010301647
4.7E−09
1.2E−08
80%
4.0E−09
8.0E−09
75%
ND

F010301648
3.4E−09
2.1E−07
30%
3.2E−09
8.9E−08
25%
ND

F010301649
4.2E−09
3.4E−09
100%
4.1E−09
5.3E−09
100%
ND

F010301849
ND
ND
ND
1.8E−09
3.0E−09
100%
ND

F010301850
3.0E−09
5.4E−09
ND
1.8E−09
2.8E−09
100%
1.9E−09

F010302307
ND
ND
ND
1.9E−09
2.8E−09
100%
2.9E−09

F010302308
ND
ND
ND
1.3E−09
1.8E−09
100%
2.0E−09

F010302309
ND
ND
ND
1.5E−09
2.2E−09
100%
2.3E−09

F010302391
2.3E−09
2.7E−09
100%
2.0E−09
1.9E−09
100%
ND

F010302392
ND
ND
ND
ND
ND
ND
ND

F0103387G05
3.6E−09
2.8E−09
100%
1.9E−09
3.1E−09
100%
2.7E−09

ND, not determined

Part 3

ID #
Tm [° C.]
aSEC
OD340 nm

F010301556
69
ND
ND

F010301563
74
ND
ND

F010301643
76
ok
ok

F010301644
71
ok
ok

F010301645
73
ok
ok

F010301646
73
ok
ok

F010301647
68
ok
ok

F010301648
66
ok
ok

F010301649
72
ok
ok

F010301849
76
ok
ok

F010301850
76
ok
ok

F010302307
76
ok
ok

F010302308
75
ok
ok

F010302309
76
ok
ok

F010302391
73
ok
ok

F010302392
ND
ND
ND

F0103387G05
74
ok
ok

ND, not determined

ok, OD360 is acceptable

Selection of an F0103387G05 Sequence Optimization Variant

Variant F010302391 was selected as the final sequence optimization variant of F0103387G05 (see F0103387G05 SO in FIG. 29). It boasts a comparable binding on human Nav1.7α, as well as comparable aSEC and OD340 nm behavior, an improved thermal stability at low pH and a reduced Tagg (Table 36 and FIG. 30). In vitro electrophysiology experiments confirmed the low nM potency on huNav1.7α and selectivity over Nav1.4α, Nav1.5α, and Nav1.6α. All PTM liabilities (Table 32) were substituted with the exception of N73. Cell-free fermentation expression titers at CMC of the corresponding monovalent tagless variant F010302400 in P. pastoris were 2.5 g/L.

TABLE 36

Sequence optimization variant of F010387G05

Part 1

HEK FlpIn

HEKa/β1

Nav157ch14-
HEK FlpIn
(SEQ ID NO:

β1-β2- β3
Nav157ch14
40) Nav1.7α
HEKa Nav1.7α

(SEQ ID NO:
(SEQ ID NO:
(SEQ ID NO:
(SEQ ID NO: 1)

ID #
20) EC50 [M]
19) EC50 [M]
1) EC50 [M]
EC50 [M]

F0103387G05
3.6E−09
2.8E−09
1.9E−09
3.1E−09

F010302391
2.3E−09
2.7E−09
2.0E−09
1.9E−09

—, no activity observed @ 7 μM

Part 2

HEK
HEK
HEK
HEK

HEK
rhNav1.7α +
huNav1.4α
huNav1.5α
huNav1.6α

huNav1.7α + β1
β1-β2-β3
(SEQ ID
(SEQ ID
(SEQ ID

(SEQ ID NO:
(SEQ ID
NO: 26)
NO: 27)
NO: 28)

ID #
44) IC50 [M]
NO: 4)
IC50
IC50
IC50

F0103387G05
ND
ND
ND
ND
ND

F010302391
3.2E−08
—
—
—
—

—, no activity observed @ 7 μM;

ND, not determined

Part 3

ID #
Tm [° C.]
Tagg [° C.]
aSEC
OD340 nm
AbM
Kabat

F0103387G05
74
>70
ok
ok
81%
76%

F010302391
73
53
ok
ok
87%
82%

ok, OD360 is acceptable

Example 10
Sequence Optimization of F0103464B09

Several PTM substitution libraries were generated based on the accelerated stress data summarized in Table 37 and screened as crude periplasmic extracts in binding FACS on human, rhesus and murine Nav1.7α. No substitution libraries were generated for M77 and N53 substitutions.

TABLE 37

Results accelerated stress experiments performed on F0103464B09 variants

Modification

ID #
Description
Site
Stress condition
observed

F0103464B09
F0103464B09-FLAG3-
NA
1 week @ 45° C., ±1
0.1% increase

HIS6

mg/mL in D-PBS
of pre-peak

(SE-HPLC)

F010301669
F0103464B09-HIS6
N53
4 weeks @ −20, 25
>20%

and 40° C.

F010301669
F0103464B09-HIS6
M77
10 mM H₂O₂for 3 h
>25%

@ RT

F010301669
F0103464B09-HIS6
N99
4 weeks @ −20, 25
>20%

and 40° C.

F010302363
F0103464B09(L11V, T24A,
NA
1 week @ 45° C., ±1
0% increase

T25S, S26H, R39Q, V40A, E44Q,

mg/mL in D-PBS
of pre-peak

N53E, N58Q, F62S, A63V,

(SE-HPLC)

S68T, M77T, T79Y, R81Q,

S82aN, N82bS, K83R, G88A,

V89L, N99S)-FLAG3-HIS6

NA, not applicable

N99S substitution resulted in a comparable or slightly improved binding profile compared to the parental reference F0103464B09 (Table 38) and was retained.

TABLE 38

Summary screening N99X & T100X substitution libraries

CHO Flp-In
CHO Flp-In

huNav1.7α +
rhNav1.7α +
HEK Jmp-In

β1-β2-β3
β1-β2-β3
muNaV1.7α

(SEQ ID NO: 3)
(SEQ ID NO: 4)
(SEQ ID NO: 1)

Mean
SD
Mean
SD
Mean
SD

MFI
MFI
MFI
MFI
MFI
MFI

Parental
51339
12286
8778
2711
10524
1686

reference

N99A
67236
10630
611
18
6953
420

N99C
2148
693
528
6
930
25

N99D
17565
2934
520
30
929
0

N99E
12920
1629
528
6
938
1

N99F
65663
17996
516
6
938
1

N99G
35986
4476
530
8
956
21

N99I
42100
3304
540
6
946
7

N99K
38359
756
537
1
923
8

N99L
35233
3218
536
10
919
19

N99M
58378
435
523
0
936
15

N99P
612
16
554
20
912
12

N99Q
44147
8475
546
10
1487
123

N99R
54569
3670
572
0
975
5

N99S
68583
1345
4867
116
18128
1883

N99T
43801
75
839
22
12506
1026

N99V
48230
6360
552
6
952
16

N99W
102535
10470
530
2
3324
3361

N99Y
50347
70381
490
20
936
61

T100A
46487
4744
14750
239
13767
654

T100D
40000
8423
526
1
1105
37

T100E
41841
2122
518
33
973
23

T100F
30496
1300
533
30
997
19

T100G
23035
1891
2835
240
6429
120

T100H
53777
5512
689
4
5147
210

T100I
35094
8735
541
13
1311
24

T100K
24860
2730
583
15
8089
123

T100L
32189
9188
531
18
1251
175

T100M
38964
6535
585
34
2629
593

T100P
16711
1179
1581
206
2092
101

T100Q
39436
9907
650
53
4295
787

T100R
30391
2087
626
49
10230
957

T100S
50304
5618
11824
3287
11225
1855

T100V
27409
1370
564
13
1680
16

T100Y
25436
1212
526
16
1121
43

Characterization of F0103464B09 Variants

In the first round, a large number of sequence optimization substitutions were explored. In the second round, eight different affinity maturation combinations (see Example 3) were explored for improved binding to rhesus Nav1.7α, combined with the remaining sequence optimization substitutions. The variants were compared in binding FACS on human, rhesus and murine Nav1.7α (muNav1.7α), in aSEC for possible for possible multimerization, in OD340 for insoluble aggregate formation, and in the thermal shift assay for Tm (Table 40). Table 35 summarizes the effects of the explored substitutions.

In the course of the sequence optimization process, subtle drops in binding to rhesus Nav1.7α were observed for the following substitutions: R39Q, A63V, T79Y, R81Q and N99S (Table 39). R39Q substitution also resulted in a subtle drop in binding to human Nav1.7α (Table 39). The combination of these, as present in the background in which the combinatorial affinity maturation substitutions were introduced, resulted in the complete abolishment of binding to rhesus Nav1.7α for the controls that do not carry any of the affinity maturation substitutions (variants F010302365, F010302366 and F010302368 in Table 40) and the same was observed for the variants combining the A28Q G54E substitutions. Less outspoken, none of the variants combining the A28Q G54E N58Q, S26H A28Q G54E N58Q or A28Q N53E G54S N58Q substitutions reached maximum binding levels to rhesus Nav1.7α (Table 40). A similar observation was made for the variants combining the S26H V33L N53E G54S substitutions, which also resulted in a drop in binding EC50 to human Nav1.7α. The three remaining combinations S26H N53E N58Q, S26H N53E G54S N58Q and S26H A28Q N53E N58Q were highly comparable for their binding to human and rhesus Nav1.7α (Table 40). The S26H N53E N58Q combination was then selected as it achieves the same binding improvements with one mutation less than the two others.

TABLE 39

Overview of F0103464B09 substitutions

huNav1.7α
rhNav1.7α
muNav1.7α

EC50 fold
EC50 fold
EC50 fold
Tm
aSEC

change
change
change
difference
behavior

compared
compared
compared
compared
compared
OD340

to
to
to
to
to
compared to

Substitution
reference
reference
reference
reference
reference
reference

L11V
=
=
−
−2
=
=

(L11V, K83R,

V89L)

T24A
=
=
−2
+1
=
=

T25S
=
=
−2
=
=
=

S26H
=
+3
ND
+3
=
=

(S26H, N53E,

N58Q)

A28Q
+
+
ND
ND
ND
ND

V33L
=
+3
ND
ND
ND
ND

R39Q
−
−
−4
+3
=
=

V40A
=
=
=
−1
=
=

E44Q
=
+3
+3
=
=
=

N53E
−
+
ND
+3
=
=

(S26H, N53E,

N58Q)

G54E
=
+
ND
ND
ND
ND

G54S
=
+
ND
ND
ND
ND

N58Q
+
+2
ND
+3
=
=

(S26H, N53E,

N58Q)

F62S
=
=
=
−8
=
=

A63V
=
−
−2
−5
=
=

+4 (F62S)

S68T
=
=
=
+2
=
=

K76N
−2
−
−4
=
=
=

M77T
=
=
=
+2
=
=

T79Y
=
−
=
+2
=
=

R81Q
=
−
=
+2
=
=

S82aN
=
=
=
=
=
=

N82bS
=
=
=
−3
=
=

K83R
=
=
=
−2
=
=

(L11V, K83R,

V89L)

G88A
=
=
=
−5
=

V89L
=
=
=
−2
=
=

(L11V, K83R,

V89L)

L93N
−
−−−
−−−
−3
=
=

N99S
=
−
=
ND
ND
ND

=, activity is equivalent reference

−, lower activity than reference

+, higher activity to reference

ND, not determined

TABLE 40

Characterization of F0103464B09 variants

Part 1

ID #
L11
T24
T25
S26H
A28Q
V33L
R39
V40
E44
N53E
G54E/S
N58Q
F62

F010301868
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301869
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301870
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301871
V
A
.
.
.
.
.
.
.
.
.
.
.

F010301872
V
.
S
.
.
.
.
.
.
.
.
.
.

F010301873
V
.
.
.
.
.
Q
.
.
.
.
.
.

F010301874
V
.
.
.
.
.
.
A
.
.
.
.
.

F010301875
V
.
.
.
.
.
.
.
.
.
.
.
S

F010301876
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301877
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301893
V
.
.
.
.
.
.
.
Q
.
.
.
.

F010301932
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301933
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301934
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301935
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301936
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301937
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301938
V
.
.
.
.
.
.
.
.
.
.
.
.

F010301939
V
.
.
.
.
.
.
.
.
.
.
.
.

F010302333
V
A
S
.
Q
.
.
A
Q
.
E
.
S

F010302334
V
A
S
.
Q
.
.
A
Q
.
E
Q
S

F010302335
V
A
S
.
Q
.
.
A
Q
E
S
Q
S

F010302336
V
A
S
H
Q
.
.
A
Q
.
E
Q
S

F010302337
V
A
S
H
Q
.
.
A
Q
E
.
Q
S

F010302338
V
A
S
H
.
.
.
A
Q
E
S
Q
S

F010302339
V
A
S
H
.
.
.
A
Q
E
.
Q
S

F010302340
V
A
S
H
.
L

A
Q
E
S
.
S

F010302341
V
A
S
.
Q
.
Q
A
Q
.
E
.
S

F010302342
V
A
S
.
Q
.
Q
A
Q
.
E
Q
S

F010302343
V
A
S
.
Q
.
Q
A
Q
E
S
Q
S

F010302344
V
A
S
H
Q
.
Q
A
Q
.
E
Q
S

F010302345
V
A
S
H
Q
.
Q
A
Q
E
.
Q
S

F010302346
V
A
S
H
.
.
Q
A
Q
E
S
Q
S

F010302347
V
A
S
H
.
.
Q
A
Q
E
.
Q
S

F010302348
V
A
S
H
.
L
Q
A
Q
E
S
.
S

F010302349
V
A
S
.
Q
.
.
A
Q
.
E
.
S

F010302350
V
A
S
.
Q
.
.
A
Q
.
E
Q
S

F010302351
V
A
S
.
Q
.
.
A
Q
E
S
Q
S

F010302352
V
A
S
H
Q
.
.
A
Q
.
E
Q
S

F010302353
V
A
S
H
Q
.
.
A
Q
E
.
Q
S

F010302354
V
A
S
H
.
.
.
A
Q
E
S
Q
S

F010302355
V
A
S
H
.
.
.
A
Q
E
.
Q
S

F010302356
V
A
S
H
.
L
.
A
Q
E
S
.
S

F010302357
V
A
S
.
Q
.
Q
A
Q
.
E
.
S

F010302358
V
A
S
.
Q
.
Q
A
Q
.
E
Q
S

F010302359
V
A
S
.
Q
.
Q
A
Q
E
S
Q
S

F010302360
V
A
S
H
Q
.
Q
A
Q
.
E
Q
S

F010302361
V
A
S
H
Q
.
Q
A
Q
E
.
Q
S

F010302362
V
A
S
H
.
.
Q
A
Q
E
S
Q
S

F010302363
V
A
S
H
.
.
Q
A
Q
E
.
Q
S

F010302364
V
A
S
H
.
L
Q
A
Q
E
S
.
S

F010302365
V
A
S
.
.
.
.
A
Q
.
.
.
S

F010302366
V
A
S
.
.
.
Q
A
Q
.
.
.
S

F010302368
V
A
S
.
.
.
Q
A
Q
.
.
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S

F0103464B09
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Part 1

ID #
A63
S68
K76
M77
T79
R81
S82a
N82b
K83
G88
V89
L93
N99

F010301868
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301869
.
T
.
T
Y
Q
N
S
R
A
L
.
.

F010301870
.
T
.
.
Y
Q
N
S
R
A
L
N
.

F010301871
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301872
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301873
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301874
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301875
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301876
V
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301877
.
T
N
.
Y
Q
N
S
R
A
L
.
.

F010301893
.
T
.
.
Y
Q
N
S
R
A
L
.
.

F010301932
.
.
.
.
.
.
.
.
R
.
L
.
.

F010301933
.
T
.
.
.
.
.
.
R
.
L
.
.

F010301934
.
.
.
T
.
.
.
.
R
.
L
.
.

F010301935
.
.
.
.
Y
.
.
.
R
.
L
.
.

F010301936
.
.
.
.
.
Q
.
.
R
.
L
.
.

F010301937
.
.
.
.
.
.
N
.
R
.
L
.
.

F010301938
.
.
.
.
.
.
.
S
R
.
L
.
.

F010301939
.
.
.
.
.
.
.
.
R
A
L
.
.

F010302333
.
.
.
T
Y
Q
N
S
R
A
L
.
S

F010302334
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302335
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302336
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302337
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302338
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302339
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302340
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302341
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302342
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302343
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302344
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302345
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302346
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302347
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302348
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302349
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302350
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302351
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302352
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302353
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302354
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302355
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302356
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302357
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302358
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302359
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302360
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302361
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302362
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302363
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302364
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302365
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302366
.
T
.
T
Y
Q
N
S
R
A
L
.
S

F010302368
V
T
.
T
Y
Q
N
S
R
A
L
.
S

F0103464B09
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.
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.
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Part 2

CHO

CHO
FlpIn

FlpIn
rhNav1.7α +

CHO FlpIn
rhNav1.7α +
β1-β2-β3

CHO FlpIn
huNav1.7α +
β1-β2- β3
(SEQ ID

huNav1.7α +
β1-β2-β3
(SEQ ID
NO: 4)
HEK JmpIn
HEK JmpIn

β1-β2-β3
(SEQ ID
NO: 3)
Bmax
muNav1.70α
muNav1.7α

(SEQ ID NO: 3)
NO: 3)
EC50
(including
(SEQ ID NO:
(SEQ ID NO:

ID #
EC50 [M]
Bmax
[M]
affmat mutations)
45) EC50 [M]
453) Bmax

F010301868
6.3E−09
98%
6.2E−08
ND
1.1E−08
ND

F010301869
7.6E−09
100%
9.6E−08
ND
1.7E−08
ND

F010301870
1.0E−08
100%
—
ND
—
ND

F010301871
5.8E−09
100%
3.7E−08
ND
2.4E−08
ND

F010301872
6.8E−09
100%
5.7E−08
ND
2.1E−08
ND

F010301873
9.0E−09
100%
1.1E−07
ND
4.2E−08
ND

F010301874
6.5E−09
100%
5.5E−08
ND
1.8E−08
ND

F010301875
6.4E−09
100%
5.2E−08
ND
1.4E−08
ND

F010301876
6.3E−09
100%
1.1E−07
ND
2.3E−08
ND

F010301877
1.3E−08
100%
9.6E−08
ND
3.9E−08
ND

F010301893
5.8E−09
100%
2.1E−08
ND
3.6E−09
ND

F010301932
4.3E−09
94%
1.7E−08
ND
4.8E−09
ND

F010301933
4.4E−09
94%
2.1E−08
ND
5.3E−09
ND

F010301934
4.3E−09
96%
2.1E−08
ND
4.5E−09
ND

F010301935
3.8E−09
94%
2.7E−08
ND
5.5E−09
ND

F010301936
4.4E−09
95%
2.6E−08
ND
4.8E−09
ND

F010301937
4.3E−09
95%
2.0E−08
ND
4.8E−09
ND

F010301938
4.8E−09
97%
2.0E−08
ND
4.7E−09
ND

F010301939
5.1E−09
96%
2.2E−08
ND
6.3E−09
ND

F010302333
4.1E−09
100%
—
—
ND
ND

F010302334
4.0E−09
100%
1.1E−08
70%
ND
ND

F010302335
4.1E−09
100%
1.3E−08
84%
ND
ND

F010302336
3.6E−09
100%
9.7E−09
79%
ND
ND

F010302337
3.9E−09
100%
6.8E−09
100%
ND
74%

F010302338
3.4E−09
100%
7.9E−09
100%
ND
ND

F010302339
4.3E−09
100%
6.8E−09
100%
8.7E−09
61%

F010302340
4.6E−09
100%
1.3E−08
96%
ND
ND

F010302341
5.3E−09
100%
—
—
ND
ND

F010302342
4.0E−09
100%
1.6E−08
61%
ND
ND

F010302343
6.4E−09
100%
1.8E−08
84%
ND
ND

F010302344
4.3E−09
100%
1.3E−08
78%
ND
ND

F010302345
4.9E−09
100%
1.2E−08
96%
ND
ND

F010302346
5.0E−09
100%
1.2E−08
99%
ND
ND

F010302347
4.6E−09
100%
9.6E−09
100%
1.4E−08
48%

F010302348
6.7E−09
100%
1.4E−08
95%
ND
ND

F010302349
4.4E−09
100%
—
—
ND
ND

F010302350
3.5E−09
100%
1.5E−08
46%
ND
ND

F010302351
5.6E−09
100%
1.5E−08
72%
ND
ND

F010302352
3.6E−09
100%
1.1E−08
60%
ND
ND

F010302353
4.2E−09
100%
7.9E−09
93%
ND
ND

F010302354
4.2E−09
100%
7.9E−09
96%
ND
ND

F010302355
3.5E−09
100%
6.8E−09
97%
2.1E−08
30%

F010302356
5.3E−09
100%
1.2E−08
81%
ND
ND

F010302357
5.3E−09
100%
—
—
ND
ND

F010302358
4.5E−09
100%
3.7E−08
37%
ND
ND

F010302359
6.4E−09
100%
2.4E−08
66%
ND
ND

F010302360
3.5E−09
100%
1.5E−08
58%
ND
ND

F010302361
4.8E−09
100%
1.1E−08
92%
ND
ND

F010302362
5.4E−09
100%
1.4E−08
89%
ND
ND

F010302363
4.2E−09
100%
9.3E−09
96%
4.8E−08
18%

F010302364
7.2E−09
100%
1.7E−08
74%
ND
ND

F010302365
3.1E−09
100%
—
—
ND
ND

F010302366
3.7E−09
100%
—
—
ND
ND

F010302368
3.9E−09
100%
—
—
ND
ND

F0103464B09
5.2E−09
99%
1.8E−08
33%
6.3E−09
74%

ND, not determined

Part 3

ID #
Tm [° C.]
aSEC
OD340 nm

F010301868
66
ok
ok

F010301869
67
ok
ok

F010301870
63
ok
ok

F010301871
67
ok
ok

F010301872
66
ok
ok

F010301873
69
ok
ok

F010301874
65
ok
ok

F010301875
58
ok
ok

F010301876
61
ok
ok

F010301877
66
ok
ok

F010301893
66
ok
ok

F010301932
64
ok
ok

F010301933
66
ok
ok

F010301934
66
ok
ok

F010301935
66
ok
ok

F010301936
66
ok
ok

F010301937
64
ok
ok

F010301938
61
ok
ok

F010301939
59
ok
ok

F010302333
ND
ND
ND

F010302334
ND
ND
ND

F010302335
ND
ND
ND

F010302336
ND
ND
ND

F010302337
ND
ND
ND

F010302338
ND
ND
ND

F010302339
63
ok
ok

F010302340
ND
ND
ND

F010302341
ND
ND
ND

F010302342
ND
ND
ND

F010302343
ND
ND
ND

F010302344
ND
ND
ND

F010302345
ND
ND
ND

F010302346
ND
ND
ND

F010302347
66
ok
ok

F010302348
ND
ND
ND

F010302349
ND
ND
ND

F010302350
ND
ND
ND

F010302351
ND
ND
ND

F010302352
ND
ND
ND

F010302353
ND
ND
ND

F010302354
ND
ND
ND

F010302355
66
ok
ok

F010302356
ND
ND
ND

F010302357
ND
ND
ND

F010302358
ND
ND
ND

F010302359
ND
ND
ND

F010302360
ND
ND
ND

F010302361
ND
ND
ND

F010302362
ND
ND
ND

F010302363
70
ok
ok

F010302364
ND
ND
ND

F010302365
60
ok
ok

F010302366
63
ok
ok

F010302368
66
ok
ok

F0103464B09
66
ok
ok

ND, not determined

Selection of an F0103464B09 Sequence Optimization Variant

Variant F010302363 was selected as the final sequence optimization variant of F0103464B09 (see F0103464B09_SO in FIG. 31). It boasts a strongly improved binding on rhesus Nav1.7α, reduced binding to muNav1.7a, as well as comparable aSEC and OD340 nm behavior and an improved thermal stability (Table 41). In vitro electrophysiology experiments confirmed the low nM potency on huNav1.7α and rhNav1.7α and selectivity over Nav1.4α, Nav1.5α, and Nav1.6α. All PTM liabilities (Table 37) were successfully substituted. Cell-free fermentation expression titers at CMC of the corresponding monovalent tagless variant F01032390 in P. pastoris were 2.0 g/L.

TABLE 41

Sequence optimization variant of F0103464B09

Part 1

CHO FlpIn

CHO FlpIn

huNav1.7α +
CHO FlpIn
rhNav1.7α +

β1-β2-β3
rhNav1.7α +
β1- β2-β3
HEK JmpIn
HEK JmpIn

(SEQ ID
β1- β2-β3
(SEQ ID NO:
muNav1.7α
muNav1.7α

NO: 3) EC50
(SEQ ID NO:
4) Bmax
(SEQ ID NO:
(SEQ ID NO:

ID #
[M]
4) EC50 [M]
(including
45) EC50 [M]
45) Bmax

F0103464B09
5.2E−09
1.8E−08
33%
6.3E−09
74%

F010302363
4.2E−09
9.3E−09
96%
4.8E−08
18%

—, no activity observed @ 7 μM

Part 2

HEK
HEK
HEK
HEK
HEK

huNav1.7α +
rhNav1.7α +
huNav1.4
huNav1.5
huNav1.6α

β1 (SEQ ID
β1-β2-β3
(SEQ ID
(SEQ ID
(SEQ ID

NO: 44) IC50
(SEQ ID NO:
NO: 26)
NO: 27)
NO: 28)

ID #
[M
4) IC50 [M]
IC50 [M]
IC50 [M]
IC50 [M]

F0103464B09
ND
ND
ND
ND
ND

F010302363
1.1E−08
3.6E−08
—
—
—

—, no activity observed @ 7 μM;

ND, not determined

Part 3

OD340

ID #
Tm [° C.]
Tagg [° C.]
aSEC
nm
AbM
Kabat

F0103464B09
66
Inconclusive
ok
ok
72%
69%

F010302363
70
71
ok
ok
85%
80%

ok, acceptable

Example 11
Identification of Anti-Navβ Subunit ISVDs

The aim of this campaign was to identify lead candidates that bind to different, non-overlapping epitopes compared to previously identified extracellular Nav1.7α binders (see previous examples). To this end, a selection and screening strategy was designed to identify lead candidates that would be able to bind in an avid fashion, when combined with a previously identified extracellular Nav1.7α binding ISVD.

Different immune repertoires were cloned downstream of an anchor building block [(F103275B05(N93R), a rhNav1.7α cross-reactive variant] separated by a long 50GS linker, resulting in bivalent phage display libraries.

Selections using high quality proteoliposome (PL) preparations or cell lines as antigen were performed on bivalent libraries derived from immunization schedules in which the animals first were repeatedly administered with different forms of full-length DNA, followed by up to four administrations with PL or membrane extract (ME), followed again by multiple administrations with different forms of full-length DNA. Crude periplasmic extracts containing bivalent ISVDs enriched by the selection process, were screened in binding FACS and competition FACS on different cell lines. Table 42 summarizes the screening data of five lead ISVD candidates F0103478E09, F0103492E09, F0103495F09, F0103500E03 and F0103505D08 (for the screening each F0103478E09, F0103492E09, F0103495F09, F0103500E03 and F0103505D08 was linked at the N-terminus to the C-terminus of an F103275B05(N93R)-50GS moiety to form a bivalent ISVD) for which the totality of the data in comparison to a control (bivalent ISVD F010300702 comprising an irrelevant anti-RSV building block linked at the N-terminus to the C-terminus of an F103275B05(N93R)-50GS moiety) suggests that they bind in an avid fashion to Nav1.7α:

- selective binding on hu & rhNav1.7α in HEK & CHO at higher levels than the average of control F010300702
- remaining binding after competition with anchor building block at higher levels than the average of control F010300702

Sequence analysis revealed that these lead candidates are unrelated and belong to different ISVD families (last column of (Table 42). Most of these lead candidates and/or related family members with high sequence similarity were identified multiple times throughout different selection and screening campaigns. Further characterization revealed that these lead candidates did not bind to Nav1.7α but instead were Navβ1 or Navβ2 binders.

TABLE 42

Overview binding and competition FACS screening of selected Navβ binders

Part 1

CHO
CHO

Flp-In
Flp-In

huNav1.5-
huNav1.5-

CHO
CHO
β1-β2-β3 in
β1-β2-β3 in

Flp-In
Flp-In
competition
competition

huNav1.7-
huNav1.5-
vs. 1 μM
vs. 1 μM

β1-β2-β3
β1-β2-β3
IRR00092
F010300703

ID #
(MFI)
(MFI)
(MFI)
(MFI)

F0103PMP478E09
82782
1079
2065
833

F0103PMP492E09
40185
559
1860
825

F0103PMP495F09
35600
483
930
645

F0103PMP500E03
22450
521
975
852

F0103PMP505D08
106009
891
964
845

F010300702 (n = 15)
1567
662
684
653

Part 2

CHO

CHO
CHO

Flp-In
CHO
Flp-In
Flp-In

huNav1.7-
Flp-In
rhNav1.7-
rhNav1.7-

β1-β2-β3 in
huNav1.7-
β1-β2-β3 in
β1-β2-β3 in

competition
β1-β2-β3 in
competition
competition

vs. 1 μM
competition
vs. 1 μM
vs. 1 μM

IRR00092
vs. 1 μM
IRR00092
F010300703

ID #
(MFI)

text missing or illegible when filed

(MFI)
(MFI)

F0103PMP478E09
136552
28773
139028
60635

F0103PMP492E09
104334
11981
111433
25186

F0103PMP495F09
93301
7161
90368
6280

F0103PMP500E03
33732
5376
96947
21477

F0103PMP505D08
95366
9240
108235
22537

F010300702 (n = 15)
4120
812
5168
721

Part 3

HEK

HEK

Flp-In
HEK
Flp-In

HEK293
huNav1.5-
Flp-In
huNav157chim14-

Flp-In
β1-β2-β3
huNav157chim14
β1-β2-β3

ID #
(MFI)
(MFI)
(MFI)
(MFI)
Family

F0103PMP478E09
1052
877
799
1234
1037

F0103PMP492E09
1205
1656
58377
132919
1044

F0103PMP495F09
1090
976
45146
127724
1040

F0103PMP500E03
1084
1106
47300
87229
1042

F0103PMP505D08
1041
1198
78390
136037
1053

F010300702 (n = 15)
1132
946
27719
15389
NA

NA, not applicable

text missing or illegible when filed

indicates data missing or illegible when filed

Binding Characterization Monovalent β-Subunit Binders

ISVD F0103240B04 was identified by means of binding ELISA as a candidate Navβ2 binder. Binding FACS (FIG. 33) and binding ELISA (FIG. 34B) experiments with purified monovalent protein suggest that F0103240B04 is indeed a potent Navβ2 binder. Five ISVDs, F0103478E09, F0103492E09, F0103495F09, F0103500E03 and F0103505D08, identified by binding and competition FACS (Table 42) were further characterized as purified monovalent protein. The combined data from the binding ELISA (FIGS. 34A-34C) and binding FACS experiments (FIGS. 35A-35D and FIGS. 36A-36E) suggest that F0103478E09 is a weak Navβ1 binder and that F0103492E09, F0103500E03, and F0103505D08 are weak Navβ2 binders. F0103495F09 was not evaluated as purified monovalent protein in the binding ELISA or binding FACS experiments using transiently transfected cells because binding FACS experiments using stable cell lines suggest that it recognizes a HEK293-specific cell background marker (See FIG. 36E). Additional competition FACS experiments with Nav1.7α-Navβ-subunit bispecific ISVDs; however, classify F103495F09 as a weak Navβ1 binder, similar to F0103478E09.

Binding ELISA

In general, 10 μg/mL of HEK huNav1.7α-Navβ1 (huNav1.7-(31) expressing cells and HEK293T null ME cells were coated in bicarbonate buffer (pH9.6) overnight at 4° C. in 384-well HB Spectraplate (catalog #6007500, Perkin Elmer). Wells were blocked with 4% Marvel in PBS. After addition of periplasmic extracts (either pen (1/5) or purified ISVD) diluted in 2% Marvel (Premier Foods Group, St Albans, UK) in PBS, FLAG3-tagged ISVD binding was detected using a mouse anti-Flag-HRP conjugate (catalog #A8592-1MG, Sigma) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3′,5,5′-tetramentylbenzidine) (catalog ##esTMB, SDT). Plates were read out on a MultiSkan device (ThermoFisher Scientific) at OD450. EC50 values were calculated using four-parameter logistic curves in GraphPad Prism7.

Alternatively, 3 μg/mL of HEK huNav1.7α-Navβ1-Navβ2-Navβ3 (huNav1.7-β1-β2-β3) cl. 11 PL was used as coated antigen in combination with detection of CMYC3-tagged ISVDs by mouse anti-c-myc biotin conjugate (catalog #MCA2200B Serotec) followed by extravidin-HRP conjugate (catalog #E2886, Sigma-Aldrich).

Example 12
Nav1.7α-Navβ Bispecific ISVDs

Bispecific leads were generated, fusing different anti-Navβ ISVDs to the C-terminus of the rhesus cross-reactive anti-Nav1.7α ISVD F103275B05(N93R) by means of a long flexible 50GS linker. The bispecifics were evaluated for their ability to compete for binding with the monovalent F0103275B05(N73R) variant to Nav1.7α in FACS experiments on different cell lines. The data shown in Table 43, FIGS. 37A-37B, and FIGS. 38A-38C reveals 10-to 1000-fold improved competition FACS IC50 values compared to the monovalent F0103275B05(N73R) control (F010300468 in table). This holds true for both Navfllbinders and Navβ2 binders on cell lines expressing the relevant counterparts. Also, stronger Navβ binders bring about greater IC50 improvements to the respective bispecifics. The monovalent Navβ binders were not able to displace F0103275B05(N73R) from Nav1.7α by themselves (FIGS. 38A-38C).

TABLE 43

Summary competition FACS of anti-Nav1.7α-Navβ bispecific ISVDs

Part 1

HEK
HEK

huNav1.7α
huNav1.7α-β1

Classification
vs. EC25 of
vs. EC25 of

2^nd
F103275B05(N93R)
F103275B05(N93R)

ID #
Description
ISVD
IC50 [M]
IC50 [M]

F010302375
F0103275B05(E1D, N93R)-50GS-
weak
1.1 × 10⁻⁰⁷
4.3 × 10⁻⁰⁹

F0103478E09(L108Q) + FLAG3-
Navβ1

HIS6
binder

F010302377
F0103275B05(E1D, N93R)-50GS-
weak
8.6 × 10⁻⁰⁸
1.4 × 10⁻⁰⁷

F0103492E09 + FLAG3-HIS6
Navβ2

binder

F010302378
F0103275B05(E1D, N93R)-50GS-
weak
8.0 × 10⁻⁰⁸
5.2 × 10⁻⁰⁹

F0103495F09 + FLAG3-HIS6
Navβ1

binder

F010302379
F0103275B05(E1D, N93R)-50GS-
weak
8.5 × 10⁻⁰⁸
1.5 × 10⁻⁰⁷

F0103500E03(P14A, L108Q) +
Navβ2

FLAG3-HIS6
binder

F010302380
F0103275B05(E1D, N93R)-50GS-
weak
6.7 × 10⁻⁰⁸
1.1 × 10⁻⁰⁷

F0103505D08(L108Q) + FLAG3-
Navβ2

HIS6
binder

F010300191
F0103275B05-50GS-F0103240B04 +
strong
6.7 × 10⁻⁰⁸
1.1 × 10⁻⁰⁷

FLAG3-HIS6
Navβ2

binder

F010300468
F0103275B05(N93R) + FLAG3-HIS6
NA
4.9 × 10⁻⁰⁸
6.7 × 10⁻⁰⁸

Part 2

HEK
CHO
CHO

FlpIn
FlpIn
FlpIn

huNav1.7α-
huNav1.7α-
rhNav1.7α-

β1-β2-β3 vs.
β1-β2-β3 vs.
β1-β2-β3 vs.

Classification
EC25 of
EC25 of
EC40 of

2^nd
F103275B05(N93R)
F103275B05(N93R)
F103275B05(N93R)

ID #
Description
ISVD
IC50 [M]
IC50 [M]
IC50 [M]

F010302375
F0103275B05(E1D, N93R)-
weak
1.0 × 10⁻⁰⁸
1.2 × 10⁻⁰⁸
6.5 × 10⁻⁰⁹

50GS-F0103478E09(L108Q) +
Navβ1

FLAG3-HIS6
binder

F010302377
F0103275B05(E1D, N93R)-
weak
5.2 × 10⁻⁰⁹
4.1 × 10⁻⁰⁹
1.2 × 10⁻⁰⁹

50GS-F0103492E09-FLAG3 +
Navβ2

HIS6
binder

F010302378
F0103275B05(E1D, N93R)-
weak
1.2 × 10⁻⁰⁸
1.0 × 10⁻⁰⁸
6.6 × 10⁻⁰⁹

50GS-F0103495F09-FLAG3 +
Navβ1

HIS6
binder

F010302379
F0103275B05(E1D, N93R)-
weak
1.6 × 10⁻⁰⁸
1.9 × 10⁻⁰⁸
4.8 × 10⁻⁰⁹

50GS-
Navβ2

F0103500E03(P14A, L108Q)-
binder

FLAG3 + HIS6

F010302380
F0103275B05(E1D, N93R)-
weak
8.7 × 10⁻⁰⁹
8.1 × 10⁻⁰⁹
2.0 × 10⁻⁰⁹

50GS-
Navβ2

F0103505D08(L108Q)-
binder

FLAG3 + HIS6

F010300191
F0103275B05-50GS-
strong
1.0 × 10⁻¹⁰
ND
ND

F0103240B04-FLAG3 +
Navβ2

HIS6
binder

F010300468
F103275B05(N93R) +
NA
9.7 × 10⁻⁰⁸
1.3 × 10⁻⁰⁷
7.7 × 10⁻⁰⁸

FLAG3-HIS6

NA, not applicable

NA, not applicable;

ND, not determined

Example 13

This example shows that in vivo performance may be enhanced by half-life extension (HLE), which may be particularly useful in therapeutic formats for chronic pain indications. Two types of HLE formats were evaluated: fusion to (i) the anti-SA building block ALB23002 or to (ii) huFc.

A number of pilot experiments were performed with the rhesus cross-reactive affinity maturation variant F010300659 of F0103275B05. The addition of ALB23002 to the C-terminus of F010300659 separated by a flexible GlySer linker resulted in a two- to five-fold drop in binding competition (Table 44) and functional (Table 45 and FIG. 7A) potency. In the presence of a saturating concentration of human SA, an additional two- to ten-fold reduction in potency was observed which appeared to be more pronounced for the shorter 9GS compared to the longer 35GS linker construct.

TABLE 44

Summary competition FACS of ALB23002 HLE Nav1.7 ISVDs in presence/absence of human SA

CHO FlpIn

CHO FlpIn

huNav1.7-β1-β2-

rhNav1.7-β1-β2-

CHO FlpIn
β3 vs. EC25

CHO FlpIn
β3 vs. EC25

huNav1.7-β1-β2-
of 275B05(N93R)

rhNav1.7-β1-β2-
of 275B05(N93R)

β3 vs. EC25
IC50 [M] +
Human
β3 vs. EC25
IC50 [M] +
Human

of 275B05(N93R)
50 μM
SA
of 275B05(N93R)
50 μM
SA

ID #
Description
IC50 [M]
human SA
ratio
IC50 [M]
human SA
ratio

F010301452
F0103275B05(S27P,
3.0 × 10⁻⁰⁸
ND
NA
1.6 × 10⁻⁰⁸
ND
NA

I28V, S50Y, N53P,

G55W, S56D, T57W,

N93R, A94W)

F010301465
F0103275B05(E1D,
9.8 × 10⁻⁰⁸
4.8 × 10⁻⁰⁷
5
6.0 × 10⁻⁰⁸
2.8 × 10⁻⁰⁷
5

S27P, I28V, S50Y,

N53P, G55W, S56D,

T57W, N93R, A94W)-

35GS-ALB23002

F010301555
F0103275B05(E1D,
1.4 × 10⁻⁰⁷
1.2 × 10⁻⁰⁶
8
9.2 × 10⁻⁰⁸
8.8 × 10⁻⁰⁷
10

S27P, 128V, S50Y,

N53P, G55W, S56D,

T57W, N93R, A94W)-

9GS-ALB23002

NA, not applicable;

ND, not determined

TABLE 45

Summary QPatch electrophysiology of ALB23002 HLE Nav1.7 ISVDs in presence/absence of human SA

HEK

HEK

HEK
rhNav1.7-β1-β2-β3 +
Human
HEK
huNav1.7-β1 +
Human

rhNav1.7-β1-β2-β3
10 μM human
SA
huNav1.7-β1
10 μM human
SA

ID #
Description
IC50 [M]
SA IC50 [M]
ratio
IC50 [M]
SA IC50 [M]
ratio

F010301452
F0103275B05(S27P,
1.8 × 10⁻⁰⁸
ND
NA
2.1 × 10⁻⁰⁸
ND
NA

I28V, S50Y,N53P,

G55W, S56D, T57W,

N93R, A94W)

F010301465
F0103275B05(E1D,
5.7 × 10⁻⁰⁸
1.0 × 10⁻⁰⁷
2
4.3 × 10⁻⁰⁸
2.8 × 10₋₀₇
7

S27P, I28V, S50Y,

N53P, G55W, S56D,

T57W, N93R, A94W)-

35GS-ALB23002

F010301555
F0103275B05(E1D,
3.5 × 10⁻⁰⁸
2.6 × 10⁻⁰⁷
7
4.7 × 10⁻⁰⁸
3.4 × 10⁻⁰⁷
7

S27P, I28V, S50Y,

N53P, G55W, S56D,

T57W, N93R, A94W)-

9GS-ALB23002

NA, not applicable;

ND, not determined

A number of huFc fusions were generated with the F0103265B04. The huFc moiety is based on hIgG1 with LALA and D265S mutations to reduce the interaction with FcγR. F0103265B04 is fused to the N-terminus of the huFc separated by a number of linkers with differing flexibilities as described elsewhere (Klein et al. Protein Eng Des Sel. 27:325-30 (2014), which is incorporated herein by reference in its entirety). Comparison of the different constructs in binding FACS revealed EC50 values comparable to monovalent F0103265B04 (Table 46), with the exception of 22ARO which suffered from a drop in potency. Interestingly, functional characterization using a single pulse electrophysiology protocol (FIG. 7A) revealed potencies highly favorable compared to monovalent F0103265B04 (last column of Table 46). Future experiments should determine whether these improvements are Fc- or linker-mediated.

TABLE 46

Summary binding FACS and Qpatch of F0103265B04-

Fc-fusions with different linkers

FACS

Description
HEK
FACS

[linker nomenclature
huNav1.7α-
HEK
Qpatch

according to Klein et al.
β1-β2-β3
huNav1.7α-
HEK

2014 Protein Eng Des Sel
cl.11
β1
huNav1.7α

ID #
27:325]
EC50 [M]
EC50 [M]
IC50 [M]

F0103265B04
F0103265B04-FLAG3-
ND
ND
1.2 × 10⁻⁰⁷

HIS6

22ARO
F0103265B04-L10
2.0 × 10⁻⁰⁸
2.9 × 10⁻⁰⁸
1.1 × 10⁻⁰⁷

GPZP-Fc

23ARO
F0103265B04-L1 hIgG-
6.0 × 10⁻⁰⁹
8.8 × 10⁻⁰⁹
3.6 × 10⁻⁰⁸

Fc

24ARO
F0103265B04-L17 GS1-
6.9 × 10⁻⁰⁹
9.2 × 10⁻⁰⁹
2.0 × 10⁻⁰⁸

Fc

25ARO
F0103265B04-L20 GS5-
6.1 × 10⁻⁰⁹
8.2 × 10⁻⁰⁹
4.7 × 10⁻⁰⁸

Fc

26ARO
F0103265B04-L3
8.5 × 10⁻⁰⁹
1.4 × 10⁻⁰⁸
4.0 × 10⁻⁰⁹

GPGcP-Fc

ND, not determined

Another set of Nav1.7 binder-Fc fusion proteins was generated, this time with a 5GS linker separating the two moieties, and tested for binding and electrophysiology (Table 47) following the protocol depicted in FIG. 7A. Here, affinity maturation variants F010300659 (derived from F0103275B05) and F010301656 (derived from F0103387G04) were compared to parental F0103275B05. Addition of the Fc moiety does not appear to have a major impact on the functional potency.

TABLE 47

Summary binding FACS and QPatch characterization of ISVD-5GS-Fc-fusions

FACS
Qpatch
Qpatch

FACS
HEK
HEK
HEK

HEK
huNav1.7α-
rhNav1.7α-
huNav1.7α-

huNav1.7α
β1
β1-β2-β3
β1

ID #
Description
EC50 [M]
EC50 [M]
IC50 [M]
IC50 [M]

F0103275B05
F0103275B05-
1.3 × 10⁻⁰⁸
1.3 × 10⁻⁰⁸
ND
ND

FLAG3 + HIS6

65ASP
F0103275B05-
2.1 × 10⁻⁰⁸
4.5 × 10⁻⁰⁸
ND
1.2 × 10⁻⁰⁷

5GS-Fc

F010300659
F0103275B05(S27P,
5.4 × 10⁻⁰⁹
1.1 × 10⁻⁰⁸
1.8 × 10⁻⁰⁷
8.5 × 10⁻⁰⁸

I28V, S50Y, N53P,

G55W, S56D, T57W,

N93R, A94W)-

FLAG3 + HIS6

66ASP
F010300659-5GS-
6.1 × 10⁻⁰⁸
4.2 × 10⁻⁰⁸
5.2 × 10⁻⁰⁸
9.2 × 10⁻⁰⁸

Fc

F010301656
F0103387G04(K33R,
4.9 × 10⁻⁰⁹
4.2 × 10⁻⁰⁹
6.0 × 10⁻⁰⁹
1.8 × 10⁻⁰⁸

S50Y, S56D, N93R)-

FLAG3 + HIS6

69AVB
F010301656-5GS-
1.1 × 10⁻⁰⁸
9.0 × 10⁻⁰⁹
8.0 × 10⁻⁰⁹
1.3 × 10⁻⁰⁸

Fc

ND, not determined

In a last experiment, the potencies of different HLE versions of the F0103387G04 affinity maturation variant F010301656 were compared in competition FACS and electrophysiology on huNav1.7 and rhNav1.7 (Table 48 and Table 49). As described above, the addition of an ALB23002 or Fc moiety as HLE has no outspoken effect on the potency. The presence of a saturating concentration of human SA results in a ±5-fold drop in the potency of the ALB23002 fusion.

TABLE 48

Summary competition FACS of different HLE versions of ′1656 in presence/absence of human SA

CHO FlpIn

CHO FlpIn

huNav1.7α-β1-

rhNav1.7α-β1-

CHO FlpIn
β2-β3 vs. EC25

CHO FlpIn
β2-β3 vs. EC25

huNav1.7α-β1-
of 275B05(N93R)

rhNav1.7α-β1-
of 275B05(N93R)

β2-β3 vs. EC25
IC50 [M] +
Human
β2-β3 vs. EC25
IC50 [M] +
Human

of 275B05(N93R)
50 μM
SA
of 275B05(N93R)
50 μM
SA

ID #
Description
IC50 [M]
human SA
ratio
IC50 [M]
human SA
ratio

F010301656
F0103387G04(K33R,
3.7 × 10⁻⁰⁸
3.9 × 10⁻⁰⁸
1
1.4 × 10⁻⁰⁸
1.6 × 10⁻⁰⁸
1

S50Y, S56D, N93R)-

FLAG3-HIS6

F010301940
F0103387G04(E1D,
3.5 × 10⁻⁰⁸
1.9 × 10⁻⁰⁷
5
1.3 × 10⁻⁰⁸
8.0 × 10⁻⁰⁸
6

K33R, S50Y, S56D,

N93R)-35GS-

ALB23002

69AVB
F010301656-
ND
ND

ND
ND

5GS-Fc

ND, not determined

TABLE 49

Summary QPatch electrophysiology of different HLE versions of F010301656 in presence/absence of human SA

HEK

HEK

HEK
rhNav1.7α-β1-β2-β3 +
Human
HEK
huNav1.7α-β1 +
Human
HEK

rhNav1.7α-β1-β2-β3
10 μM human
SA
huNav1.7α-β1
10 □ M human
SA
ratNav1.7α

ID #
Description
IC50 [M]
SA IC50 [M]
ratio
IC50 [M]
SA IC50 [M]
ratio
IC50 [M]

F010301656
F0103387G04
6.0 × 10⁻⁰⁹
ND
ND
1.8 × 10⁻⁰⁸
ND
ND
ND

(K33R, S50Y,

S56D, N93R)-

FLAG3-HIS6

F010301940
F0103387G04
1.6 × 10⁻⁰⁸
5.0 × 10⁻⁰⁸
3
4.6 × 10⁻⁰⁸
1.5 × 10⁻⁰⁷
3
3.7 × 10⁻⁰⁸

(E1D, K33R,

S50Y, S56D,

N93R)-35GS-

ALB23002

69AVB
F010301656-
8.0 × 10⁻⁰⁹
ND
ND
1.3 × 10⁻⁰⁸
2.6 × 10⁻⁰⁸
2
2.1 × 10⁻⁰⁸

5GS-Fc

ND, not determined

Example 14
Electrophysiological Characterization of Nav1.7a Selective ISVDs on the Automated Patch Clamp System QPatch.

Whole-cell currents were measured from cells stably expressing human, rhesus, or rat Nav1.7α, 1.6α, 1.5α, 1.4α channels using the QPatch HT™ (Sophion Bioscience). Cells were grown to 60-70% confluence in T175 cell culture flasks. Cells were lifted with Accutase™ and single cell suspensions generated with two million cells/mL.

Experiments were performed at room temperature (25-29° C.). Human and rhesus Nav1.7α currents were measured holding cells −85 mV and applying 30 ms test pulses to −20 mV at a frequency 0.1 Hz. Rat Nav1.7α currents were measured holding cells at −75 mV applying 30 ms test pulses to −20 mV at a frequency 0.1 Hz. Human and rhesus Nav1.6α, Nav1.5α, and Nav1.4α were held at −85 mV, −95 mV and −80 mV, respectively. The following solutions were used: Internal Solution (in mM): 30 CsCl, 5 HEPES, 10 EGTA, 120 CsF, 5 NaF, 2 MgCl₂, pH=7.3 with CsOH; External solutions (in mM) for human and rhesus Nav1.7α: 40 NaCl, 120 NMDG, 1 KCl, 0.5 MgCl₂, 5 HEPES, 2.7 CaCl₂, pH to 7.3 with NaOH; for rat Nav1.7α: 150 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 12 Dextrose, pH 7.3 with NaOH. Sodium currents were monitored for at least five minutes in vehicle before addition of test articles. Double additions of test article were made to QPlate™ wells to achieve equilibrium. Current inhibition was measured after 60 pulses in test article. ProTX-II was used as positive control.

IC50 values, based on three concentrations, were calculated using a built-in four parameter logistic function (Hill equation): f(x)=I_min+(I_max−I_min)/(1±(IC50/[x])^h); I_min=minimal current (fixed to 0); I_max=maximal current (fixed to a value of 100); IC50=half maximal inhibitory concentration; h=Hill coefficient.

Table 50, Table 51, Table 52, Table 53, Table 54, and Table 55 show the results. In Tables 50-55, N.E. means “no effect” and ND means “not determined”.

TABLE 50

Qpatch IC50s (nM) of Parental clones

Part 1

Human

Rhesus

Nav1.7α +
Human
Human
Human
Nav1.7α +

ID #
β1
Nav1.6α
Nav1.5α
Nav1.4α
β1-β2-β3

F0103265B04
120
N.E. @14.5 μM
ND
ND
ND

F0103362B08
1
ND
ND
ND
N.E.

F0103454D07
38
ND
ND
ND
ND

F010346B09
7
ND
ND
ND
166

Part 2

Rhesus
Rhesus
Rhesus
Rat

ID #
Nav1.6α
Nav1.5α
Nav1.4α
Nav1.7α

F0103265B04
ND
ND
ND
ND

F0103362B08
ND
ND
ND
ND

F0103454D07
ND
ND
ND
ND

F0103464B09
ND
ND
ND
ND

TABLE 51

Qpatch IC50s (nM) of F0103275B05 and F0103387G05 affinity-matured variants

Part 1

Human

Rhesus

Nav1.7α +
Human
Human
Human
Nav1.7α +

ID #
β1
Nav1.6α
Nav1.5α
Nav1.4α
β1-β2-β3

F10301656*
13
N.E.
N.E.
N.E.
8

@30 μM
@30 μM
@30 μM

F010302383
22
ND
ND
ND
3

F010300659
85
N.E.
ND
ND
199

@6.3 μM

F010300880
27
ND
ND
ND
122

F010300900
196
ND
ND
ND
36

F010300948
315
ND
ND
ND
58

F010300990
249
ND
ND
ND
101

F010300477
96
ND
ND
ND
1192

F010300631
73
ND
ND
ND
173

F010300684
202
ND
ND
ND
212

Part 2

Rhesus
Rhesus
Rhesus
Rat

ID #
Nav1.6α
Nav1.5α
Nav1.4α
Nav1.7α

F10301656*
N.E. @30 μM
N.E. @30 μM
N.E. @30 μM
21

F010302383
N.E. @7 μM
N.E. @7 μM
N.E. @7 μM
ND

F010300659
ND
ND
ND
ND

F010300880
ND
ND
ND
ND

F010300900
ND
ND
ND
ND

F010300948
ND
ND
ND
ND

F010300990
ND
ND
ND
ND

F010300477
ND
ND
ND
ND

F010300631
ND
ND
ND
ND

F010300684
ND
ND
ND
ND

*ISVD with human IgG1 Fc

TABLE 52

Qpatch IC50s (nM) of F0103265A11 affinity-matured variants

Part 1

Human

Rhesus

Nav1.7α +
Human
Human
Human
Nav1.7α +

ID #
β1
Nav1.6α
Nav1.5α
Nav1.4α
β1-β2-β3

F010301162
32
ND
ND
ND
ND

F010301191
63
ND
ND
ND
ND

F010301080
14
ND
ND
ND
ND

F010301090
22
ND
ND
ND
ND

F010301129
126
ND
ND
ND
ND

Part 2

Rhesus
Rhesus
Rhesus
Rat

ID #
Nav1.6α
Nav1.5α
Nav1.4α
Nav1.7α

F010301162
ND
ND
ND
ND

F010301191
ND
ND
ND
ND

F010301080
ND
ND
ND
ND

F010301090
ND
ND
ND
ND

F010301129
ND
ND
ND
ND

TABLE 53

Qpatch IC50s (nM) of F0103387G05 affinity-matured variants

Part 1

Human

Rhesus

Nav1.7α +
Human
Human
Human
Nav1.7α +

ID #
β1
Nav1.6α
Nav1.5α
Nav1.4α
β1-β2-β3

F010301558
8
ND
ND
ND
ND

F010301559
12
ND
ND
ND
ND

F010301563
5
ND
ND
ND
ND

F010301566
18
ND
ND
ND
ND

F010302391
32
ND
ND
ND
ND

Part 2

Rhesus
Rhesus
Rhesus
Rat

ID #
Nav1.6α
Nav1.5α
Nav1.4α
Nav1.7α

F010301558
ND
ND
ND
ND

F010301559
ND
ND
ND
ND

F010301563
ND
ND
ND
ND

F010301566
ND
ND
ND
ND

F010302391
N.E. @7 μM
N.E. @7 μM
N.E. @7 μM
N.E. @7 μM

TABLE 54

Qpatch IC50s (nM) of F0103464B09 affinity-matured variants

Part 1

Human

Rhesus

Nav1.7α +
Human
Human
Human
Nav1.7α +

ID #
β1
Nav1.6α
Nav1.5α
Nav1.4α
β1-β2-β3

F010302363
7
ND
ND
ND
166

Part 2

Rhesus
Rhesus
Rhesus
Rat

ID #
Nav1.6α
Nav1.5α
Nav1.4α
Nav1.7α

F010302363
N.E. @7 μM
N.E. @7 μM
N.E. @7 μM
N.E. @7 μM

TABLE 55

Qpatch IC50s (nM) of anti-Nav1.7α-Navβ bispecific ISVDs

Part 1

Human
Human

Human
Nav1.7α +
Nav1.2α +

ID #
Description
Nav1.7α
β1-β2-β3
β1-β2

F010300468
F0103275B05(N93R)
57
99
ND

F010302375
F0103275B05(E1D, N93R)-50GS-
123
2.6
N.E.

F0103478E09(L108Q)-FLAG3-

HIS6

F010302378
F0103275B05(E1D, N93R)-50GS-
115
2.3
N.E.

F0103495F09-FLAG3-HIS6

F010302377
F0103275B05(E1D, N93R)-50GS-
74
0.8
ND

F0103492E09-FLAG3-HIS6

F010302379
F0103275B05(E1D, N93R)-50GS-
111
3.4
ND

F0103500E03(P14A, L108Q)-

FLAG3-HIS6

Part 2

Rhesus

Rhesus
Nav1.7α +

ID #
Description
Nav1.7α
β1-β2-β3

F010300468
F0103275B05(N93R)
ND
ND

F010302375
F0103275B05(E1D, N93R)-50GS-
93
2.8

F0103478E09(L108Q)-FLAG3-

HIS6

F010302378
F0103275B05(E1D, N93R)-50GS-
103
2.6

F0103495F09-FLAG3-HIS6

F010302377
F0103275B05(E1D, N93R)-50GS-
104
75

F0103492E09-FLAG3-HIS6

F010302379
F0103275B05(E1D, N93R)-50GS-
133
131

F0103500E03(P14A, L108Q)-

FLAG3-HIS6

The amino acid and nucleotide sequences for the Nav1.7 binders, CDRs, and other molecules disclosed herein are set forth Table 56.

TABLE 56

Table of Sequences

SEQ

ID

NO:
Description
Sequence

1
huNav1.7 (alpha-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

subunit)
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKK

2
rhNav1.7 (alpha-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

subunit)
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKK

3
huNav1.7-beta1-beta2-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta3 viral P2A
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

sequences italics; beta1-
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

beta2-beta3 are in bold
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKSGRGSGATNFSLLKQAGDVEENPGPMGRLLA

LVVGAALVSSACGGCVEVDSETEAVYGMTFKIL

CISCKRRSETNAETFTEWTFRQKGTEEFVKILRY

ENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSI

FITNVTYNHSGDYECHVYRLLFFENYEHNTSVV

KKIHIEVVDKANRDMASIVSEIMMYVLIVVLTIW

LVAEMIYCYKKIAAATETAAQENASEYLAITSES

KENCTGVQVAE
GSGATNFSLLKQAGDVEENPGP
M

HRDAWLPRPAFSLTGLSLFFSLVPPGRSMEVTVP

ATLNVLNGSDARLPCTFNSCYTVNHKQFSLNWT

YQECNNCSEEMFLQFRMKIINLKLERFQDRVEF

SGNPSKYDVSVMLRNVQPEDEGIYNCYIMNPPD

RHRGHGKIHLQVLMEEPPERDSTVAVIVGASVG

GFLAVVILVLMVVKCVRRKKEQKLSTDDLKTEE

EGKTDGEGNPDDGAK
GSGATNFSLLKQAGDVEEN

PGP
MPAFNRLFPLASLVLIYWVSVCFPVCVEVPS

ETEAVQGNPMKLRCISCMKREEVEATTVVEWF

YRPEGGKDFLIYEYRNGHQEVESPFQGRLQWN

GSKDLQDVSITVLNVTLNDSGLYTCNVSREFEFE

AHRPFVKTTRLIPLRVTEEAGEDFTSVVSEIMMY

ILLVFLTLWLLIEMIYCYRKVSKAEEAAQENASD

YLAIPSENKENSAVPVEE

4
rhNav1.7-beta1-beta2-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta3 viral P2A
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

sequences italics; beta1-
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

beta2-beta3 are in bold
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMS

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFIVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCVQNSLVNNETLESI

MNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQC

PEGYTCMKIGRNPDYGYTSFDTFSWAFLALFRLMT

QDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLI

NLILAVVAMAYEEQNQANIEEAKQKELEFQQMLD

RLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSET

SKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKL

SKSDSEENIRRKSFHLGVEGHRRAHEKRLSTPSQSP

LSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFADD

EHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPI

LPVNGKMHSAVDCNGVVSLVDGRSALMLPNGQLL

PEGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMS

RASILTNTVEELEESRQKCPPWWYRFAHKFLIWNC

SPYWIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAM

EHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAM

DPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVL

RSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLT

LVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLP

RWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAG

QAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDN

LTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFILK

TFSKKPKISREIRQTEDLNTKKENYISNYTLAEMSK

GHNFLKEKDKISGFGSCVDKYLMEDSDGQSFIHNP

SLTVTVPIAPGESDLENMNTEELSSDSDSEYSKVRL

NQSSSSECSTVDNPLPGEGEEAEAEPMNSDEPEACF

TDGCVRRFSCCQVNIESGKGKIWWNIRKTCYKIVE

HSWFESFIVLMILLSSGALAFEDIYIERKKTIKIILEY

ADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWLDF

LIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPLR

ALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFS

IMGVNLFAGKFYECINTTDGSRFPASQVPNRSECFA

LMNVSQNVRWKNLKVNFDNVGLGYLSLLQVATF

KGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVIF

IIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQ

KKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVT

NQAFDISIMVLICLNMVTMMVEKEGQSPYMTDVL

YWINVVFIILFTGECVLKLISLRYYYFTIGWNIFDFV

VVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRL

VKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYA

IFGMSNFAYVKKEDGINDMFNFETFGNSMICLFQIT

TSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDC

GNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVATE

ESTEPLSEDDFEMFYEVWEKFDPDATQFIEYNKLSD

FAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCL

DILFAFTKRVLGESGEMDSLRSQMEERFMSANPSK

VSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNV

KNISSIYIKDGDRDDDLLNKKDMAFDNVNENSSPE

KTDATSSTTSPPSYDSVTKPDKEKYEQDRTEKEDK

GKDSKESKKSGRGSGATNFSLLKQAGDVEENPGPM

GRLLALVVGAALVSSACGGCVEVDSETEAVYG

MTFKILCISCKRRSETNAETFTEWTFRQKGTEEF

VKILRYENEVLQLEEDERFEGRVVWNGSRGTKD

LQDLSIFITNVTYNHSGDYECHVYRLLFFENYEH

NTSVVKKIHIEVVDKANRDMASIVSEIMMYVLIV

VLTIWLVAEMIYCYKKIAAATETAAQENASEYL

AITSESKENCTGVQVAE
GSGATNFSLLKQAGDVEE

NPGP
MHRDAWLPRPAFSLTGLSLFFSLVPPGRS

MEVTVPATLNVLNGSDARLPCTFNSCYTVNHKQ

FSLNWTYQECNNCSEEMFLQFRMKIINLKLERF

QDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNCYI

MNPPDRHRGHGKIHLQVLMEEPPERDSTVAVIV

GASVGGFLAVVILVLMVVKCVRRKKEQKLSTD

DLKTEEEGKTDGEGNPDDGAK
GSGATNFSLLKQA

GDVEENPGP
MPAFNRLFPLASLVLIYWVSVCFPV

CVEVPSETEAVQGNPMKLRCISCMKREEVEATT

VVEWFYRPEGGKDFLIYEYRNGHQEVESPFQGR

LQWNGSKDLQDVSITVLNVTLNDSGLYTCNVSR

EFEFEAHRPFVKTTRLIPLRVTEEAGEDFTSVVS

EIMMYILLVFLTLWLLIEMIYCYRKVSKAEEAA

QENASDYLAIPSENKENSAVPVEE

5
huNav1.7(F276V)-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta1-beta2-beta3 viral
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

P2A sequences italics;
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

beta1-beta2-beta3 are in
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

bold
NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCVRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKENLYFQGDYKDHDGDYKDHDIDYKDDDDK

HHHHHHHHHHGSGATNFSLLKQAGDVEENPGPMG

RLLALVVGAALVSSACGGCVEVDSETEAVYGMT

FKILCISCKRRSETNAETFTEWTFRQKGTEEFVK

ILRYENEVLQLEEDERFEGRVVWNGSRGTKDLQ

DLSIFITNVTYNHSGDYECHVYRLLFFENYEHNT

SVVKKIHIEVVDKANRDMASIVSEIMMYVLIVVL

TIWLVAEMIYCYKKIAAATETAAQENASEYLAIT

SESKENCTGVQVAE
GSGATNFSLLKQAGDVEENPG

P
MHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEV

TVPATLNVLNGSDARLPCTFNSCYTVNHKQFSL

NWTYQECNNCSEEMFLQFRMKIINLKLERFQDR

VEFSGNPSKYDVSVMLRNVQPEDEGIYNCYIMN

PPDRHRGHGKIHLQVLMEEPPERDSTVAVIVGA

SVGGFLAVVILVLMVVKCVRRKKEQKLSTDDLK

TEEEGKTDGEGNPDDGAK
GSGATNFSLLKQAGDV

EENPGP
MPAFNRLFPLASLVLIYWVSVCFPVCVE

VPSETEAVQGNPMKLRCISCMKREEVEATTVVE

WFYRPEGGKDFLIYEYRNGHQEVESPFQGRLQ

WNGSKDLQDVSITVLNVTLNDSGLYTCNVSREF

EFEAHRPFVKTTRLIPLRVTEEAGEDFTSVVSEI

MMYILLVFLTLWLLIEMIYCYRKVSKAEEAAQE

NASDYLAIPSENKENSAVPVEE

6
huNav1.7(R277Q)-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta1-beta2-beta3 viral
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

P2A sequences italics;
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

beta1-beta2-beta3 are in
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

bold
NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFQNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKENLYFQGDYKDHDGDYKDHDIDYKDDDDK

HHHHHHHHHHGSGATNFSLLKQAGDVEENPGPMG

RLLALVVGAALVSSACGGCVEVDSETEAVYGMT

FKILCISCKRRSETNAETFTEWTFRQKGTEEFVK

ILRYENEVLQLEEDERFEGRVVWNGSRGTKDLQ

DLSIFITNVTYNHSGDYECHVYRLLFFENYEHNT

SVVKKIHIEVVDKANRDMASIVSEIMMYVLIVVL

TIWLVAEMIYCYKKIAAATETAAQENASEYLAIT

SESKENCTGVQVAE
GSGATNFSLLKQAGDVEENPG

P
MHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEV

TVPATLNVLNGSDARLPCTFNSCYTVNHKQFSL

NWTYQECNNCSEEMFLQFRMKIINLKLERFQDR

VEFSGNPSKYDVSVMLRNVQPEDEGIYNCYIMN

PPDRHRGHGKIHLQVLMEEPPERDSTVAVIVGA

SVGGFLAVVILVLMVVKCVRRKKEQKLSTDDLK

TEEEGKTDGEGNPDDGAK
GSGATNFSLLKQAGDV

EENPGP
MPAFNRLFPLASLVLIYWVSVCFPVCVE

VPSETEAVQGNPMKLRCISCMKREEVEATTVVE

WFYRPEGGKDFLIYEYRNGHQEVESPFQGRLQ

WNGSKDLQDVSITVLNVTLNDSGLYTCNVSREF

EFEAHRPFVKTTRLIPLRVTEEAGEDFTSVVSEI

MMYILLVFLTLWLLIEMIYCYRKVSKAEEAAQE

NASDYLAIPSENKENSAVPVEE

7
huNav1.7(E281V)-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta1-beta2-beta3
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

viral P2A sequences
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

italics; beta1-beta2-
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

beta3 are in bold
NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLVNNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKENLYFQGDYKDHDGDYKDHDIDYKDDDDK

HHHHHHHHHHGSGATNFSLLKQAGDVEENPGPMG

RLLALVVGAALVSSACGGCVEVDSETEAVYGMTF

KILCISCKRRSETNAETFTEWTFRQKGTEEFVKILRY

ENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSIFIT

NVTYNHSGDYECHVYRLLFFENYEHNTSVVKKIHI

EVVDKANRDMASIVSEIMMYVLIVVLTIWLVAEMI

YCYKKIAAATETAAQENASEYLAITSESKENCTGV

QVAEGSGATNFSLLKQAGDVEENPGPMHRDAWLP

RPAFSLTGLSLFFSLVPPGRSMEVTVPATLNVLN

GSDARLPCTFNSCYTVNHKQFSLNWTYQECNNC

SEEMFLQFRMKIINLKLERFQDRVEFSGNPSKYD

VSVMLRNVQPEDEGIYNCYIMNPPDRHRGHGKI

HLQVLMEEPPERDSTVAVIVGASVGGFLAVVILV

LMVVKCVRRKKEQKLSTDDLKTEEEGKTDGEG

NPDDGAK
GSGATNFSLLKQAGDVEENPGP
MPAFNR

LFPLASLVLIYWVSVCFPVCVEVPSETEAVQGNP

MKLRCISCMKREEVEATTVVEWFYRPEGGKDF

LIYEYRNGHQEVESPFQGRLQWNGSKDLQDVSI

TVLNVTLNDSGLYTCNVSREFEFEAHRPFVKTTR

LIPLRVTEEAGEDFTSVVSEIMMYILLVFLTLWL

LIEMIYCYRKVSKAEEAAQENASDYLAIPSENKE

NSAVPVEE

8
huNav1.7(V331M)-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

beta1-beta2-beta3 viral
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

P2A sequences italics;
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

beta1-beta2-beta3 are in
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

bold
NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCMKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKENLYFQGDYKDHDGDYKDHDIDYKDDDDK

HHHHHHHHHHGSGATNFSLLKQAGDVEENPGPMG

RLLALVVGAALVSSACGGCVEVDSETEAVYGMT

FKILCISCKRRSETNAETFTEWTFRQKGTEEFVK

ILRYENEVLQLEEDERFEGRVVWNGSRGTKDLQ

DLSIFITNVTYNHSGDYECHVYRLLFFENYEHNT

SVVKKIHIEVVDKANRDMASIVSEIMMYVLIVVL

TIWLVAEMIYCYKKIAAATETAAQENASEYLAIT

SESKENCTGVQVAE
GSGATNFSLLKQAGDVEENPG

P
MHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEV

TVPATLNVLNGSDARLPCTFNSCYTVNHKQFSL

NWTYQECNNCSEEMFLQFRMKIINLKLERFQDR

VEFSGNPSKYDVSVMLRNVQPEDEGIYNCYIMN

PPDRHRGHGKIHLQVLMEEPPERDSTVAVIVGA

SVGGFLAVVILVLMVVKCVRRKKEQKLSTDDLK

TEEEGKTDGEGNPDDGAK
GSGATNFSLLKQAGDV

EENPGP
MPAFNRLFPLASLVLIYWVSVCFPVCVE

VPSETEAVQGNPMKLRCISCMKREEVEATTVVE

WFYRPEGGKDFLIYEYRNGHQEVESPFQGRLQ

WNGSKDLQDVSITVLNVTLNDSGLYTCNVSREF

EFEAHRPFVKTTRLIPLRVTEEAGEDFTSVVSEI

MMYILLVFLTLWLLIEMIYCYRKVSKAEEAAQE

NASDYLAIPSENKENSAVPVEE

9
huNav1.7(N146S, V194I,
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

F276V, R277Q, E281V,
EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

V331M, E504D, D507E,
EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

S508N, N533S)-beta1-
LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMS

beta2-beta3 viral P2A
NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

sequences italics; beta1-
FLRDPWNWLDFIVIVFAYLTEFVNLGNVSALRTFR

beta2-beta3 are in bold
VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCVQNSLVNNETLESI

MNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQC

PEGYTCMKIGRNPDYGYTSFDTFSWAFLALFRLMT

QDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLI

NLILAVVAMAYEEQNQANIEEAKQKELEFQQMLD

RLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSET

SKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKL

SKSDSEENIRRKSFHLGVEGHRRAHEKRLSTPSQSP

LSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFADD

EHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPP

MLPVNGKMHSAVDCNGVVSLVDGRSALMLPNGQ

LLPEGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRA

MSRASILTNTVEELEESRQKCPPWWYRFAHKFLIW

NCSPYWIKFKKCIYFIVMDPFVDLAITICIVLNTLFM

AMEHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLI

AMDPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLS

VLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGN

LTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCT

LPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEV

AGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSS

DNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFI

LKAFSKKPKISREIRQAEDLNTKKENYISNHTLAEM

SKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIH

NPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKV

RLNRSSSSECSTVDNPLPGEGEEAEAEPMNSDEPEA

CFTDGCVRRFSCCQVNIESGKGKIWWNIRKTCYKI

VEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIIL

EYADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWL

DFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRP

LRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWL

IFSIMGVNLFAGKFYECINTTDGSRFPASQVPNRSEC

FALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVA

TFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYF

VVFIIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFM

TEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIF

DLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHM

TEVLYWINVVFIILFTGECVLKLISLRHYYFTVGWNI

FDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIG

RILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLV

MFIYAIFGMSNFAYVKKEDGINDMFNFETFGNSMI

CLFQITTSAGWDGLLAPILNSKPPDCDPKKVHPGSS

VEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILEN

FSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFIE

FSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSG

DRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMS

ANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRL

RQNVKNISSIYIKDGDRDDDLLNKKDMAFDNVNEN

SSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRTEK

EDKGKDSKESKKSGRGSGATNFSLLKQAGDVEENPG

P
MGRLLALVVGAALVSSACGGCVEVDSETEAVY

GMTFKILCISCKRRSETNAETFTEWTFRQKGTE

EFVKILRYENEVLQLEEDERFEGRVVWNGSRGT

KDLQDLSIFITNVTYNHSGDYECHVYRLLFFENY

EHNTSVVKKIHIEVVDKANRDMASIVSEIMMYV

LIVVLTIWLVAEMIYCYKKIAAATETAAQENASE

YLAITSESKENCTGVQVAE
GSGATNFSLLKQAGDV

EENPGP
MHRDAWLPRPAFSLTGLSLFFSLVPPGR

SMEVTVPATLNVLNGSDARLPCTFNSCYTVNHK

QFSLNWTYQECNNCSEEMFLQFRMKIINLKLER

FQDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNC

YIMNPPDRHRGHGKIHLQVLMEEPPERDSTVAV

IVGASVGGFLAVVILVLMVVKCVRRKKEQKLST

DDLKTEEEGKTDGEGNPDDGAK
GSGATNFSLLK

QAGDVEENPGP
MPAFNRLFPLASLVLIYWVSVCF

PVCVEVPSETEAVQGNPMKLRCISCMKREEVEA

TTVVEWFYRPEGGKDFLIYEYRNGHQEVESPFQ

GRLQWNGSKDLQDVSITVLNVTLNDSGLYTCNV

SREFEFEAHRPFVKTTRLIPLRVTEEAGEDFTSV

VSEIMMYILLVFLTLWLLIEMIYCYRKVSKAEEA

AQENASDYLAIPSENKENSAVPVEE

10
huNav157 chimera 1
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPLNKYQGFIFDIVTKQ

AFDVTIMFLICLNMVTMMVETDDQSPEKINILAKIN

LLFVAIFTGECIVKLAALRHYYFTNSWNIFDFVVVI

LSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRILRLIRG

AKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGM

ANFAYVKWEAGIDDMFNFQTFANSMLCLFQITTSA

GWDGLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAV

GILFFTTYIIISFLIVVNMYIAIILENFSVATEESTEPLS

EDDFDMFYEIWEKFDPEATQFIEYSVLSDFADALSE

PLRIAKPNQISLINMDLPMVSGDRIHCMDILFAFTKR

VLGESGEMDALKIQMEEKFMAANPSKISYEPITTTL

RRKHEEVSAMVIQRAFRRHLLQRSLKHASFLFRQQ

AGSGLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSI

SSTSFPPSYDSVTRATSDNLQVRGSDYSHSEDLADF

PPSPDRDRESIV

11
huNav157 chimera 2
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSADNLT

APDEDREMNNLQLALARIQRGLRFVKRTTWDFCC

GLLRQRPQKPAALAAQGQLPSCIATPYSPPPPETEK

VPPTRKETRFEEGEQPGQGTPGDPEPVCVPIAVAES

DTDDQEEDEENSLGTEEESSKQESQPVSGGPEAPPD

SRTWSQVSATASSEAEASASQADWRQQWKAEPQA

PGCGETPEDSCSEGSTADMINTAELLEQIPDLGQDV

KDPEDCFTEGCVRRCPCCAVDTTQAPGKVWWRLR

KTCYHIVEHSWFETFIIFMILLSSGALAFEDIYLEER

KTIKVLLEYADKMFTYVFVLEMLLKWVAYGFKKY

FTNAWCWLDFLIVDVSLVSLVANTLGFAEMGPIKS

LRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMN

VLLVCLIFWLIFSIMGVNLFAGKFGRCINQTEGDLP

LNYTIVNNKSQCESLNLTGELYWTKVKVNFDNVG

AGYLALLQVATFKGWMDIMYAAVDSRGYEEQPQ

WEYNLYMYIYFVIFIIFGSFFTLNLFIGVIIDNFNQQK

KKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIP

RPGNKIQGCIFDLVTNQAFDISIMVLICLNMVTMMV

EKEGQSQHMTEVLYWINVVFIILFTGECVLKLISLR

HYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPT

LFRVIRLARIGRILRLVKGAKGIRTLLFALMMSLPA

LFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMF

NFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDC

DPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVV

NMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEK

FDPDATQFIEFSKLSDFAAALDPPLLIAKPNKVQLIA

MDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLR

SQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVI

QRAYRRYRLRQNVKNISSIYIKDGDRDDDLLNKKD

MAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKE

KYEQDRTEKEDKGKDSKESKK

12
huNav157 chimera 3
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQATIAETEEKEKRFQEAMEMLK

KEHEALTIRGVDTVSRSSLEMSPLAPVNSHERRSKR

RKRMSSGTEECGEDRLPKSDSEDGPRAMNHLSLTR

GLSRTSMKPRSSRGSIFTFRRRDLGSEADFADDENS

TAGESESHHTSLLVPWPLRRTSAQGQPSPGTSAPGH

ALHGKKNSTVDCNGVVSLLGAGDPEATSPGSHLLR

PVMLEHPPDTTTPSEEPGGPQMLTSQAPCVDGFEEP

GARQRALSAVSVLTSALEELEESRHKCPPCWNRLA

QRYLIWECCPLWMSIKQGVKLVVMDPFTDLTITMC

IVLNTLFMALEHYNMTSEFEEMLQVGNLVFTGIFT

AEMTFKIIALDPYYYFQQGWNIFDSIIVILSLMELGL

SRMSNLSVLRSFRLLRVFKLAKSWPTLNTLIKIIGNS

VGALGNLTLVLAIIVFIFAVVGMQLFGKNYSELRDS

DSGLLPRWHMMDFFHAFLIIFRILCGEWIETMWDC

MEVSGQSLCLLVFLLVMVIGNLVVLNLFLALLLSSF

SSDNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLR

EFILKAFSKKPKISREIRQAEDLNTKKENYISNHTLA

EMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSF

IHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSK

VRLNRSSSSECSTVDNPLPGEGEEAEAEPMNSDEPE

ACFTDGCVRRFSCCQVNIESGKGKIWWNIRKTCYK

IVEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIIL

EYADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWL

DFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRP

LRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWL

IFSIMGVNLFAGKFYECINTTDGSRFPASQVPNRSEC

FALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVA

TFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYF

VVFIIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFM

TEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIF

DLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHM

TEVLYWINVVFIILFTGECVLKLISLRHYYFTVGWNI

FDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIG

RILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLV

MFIYAIFGMSNFAYVKKEDGINDMFNFETFGNSMI

CLFQITTSAGWDGLLAPILNSKPPDCDPKKVHPGSS

VEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILEN

FSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFIE

FSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSG

DRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMS

ANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRL

RQNVKNISSIYIKDGDRDDDLLNKKDMAFDNVNEN

SSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRTEK

EDKGKDSKESKK

13
huNav157 chimera 4
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGN

VSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIR

RSRIMGLSESSSETSKLSSKSAKERRNRRKKKNQKK

LSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEGHRR

AHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGR

GRDIGSETEFADDEHSIFGDNESRRGSLFVPHRPQE

RRSSNISQASRSPPMLPVNGKMHSAVDCNGVVSLV

DGRSALMLPNGQLLPEGTTNQIHKKRRCSSYLLSE

DMLNDPNLRQRAMSRASILTNTVEELEESRQKCPP

WWYRFAHKFLIWNCSPYWIKFKKCIYFIVMDPFVD

LAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNLVF

TGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLS

LVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNM

LIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKS

YKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCG

EWIETMWDCMEVAGQAMCLIVYMMVMVIGNLVV

LNLFLALLLSSFSSDNLTAIEEDPDANNLQIAVTRIK

KGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTK

KENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDK

HLMEDSDGQSFIHNPSLTVTVPIAPGESDLENMNAE

ELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEE

AEAEPMNSDEPEACFTDGCVRRFSCCQVNIESGKG

KIWWNIRKTCYKIVEHSWFESFIVLMILLSSGALAF

EDIYIERKKTIKIILEYADKIFTYIFILEMLLKWIAYG

YKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDL

GPIKSLRTLRALRPLRALSRFEGMRVVVNALIGAIPS

IMNVLLVCLIFWLIFSIMGVNLFAGKFYECINTTDGS

RFPASQVPNRSECFALMNVSQNVRWKNLKVNFDN

VGLGYLSLLQVATFKGWTIIMYAAVDSVNVDKQP

KYEYSLYMYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQ

KKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPI

PRPGNKIQGCIFDLVTNQAFDISIMVLICLNMVTMM

VEKEGQSQHMTEVLYWINVVFIILFTGECVLKLISL

RHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSP

TLFRVIRLARIGRILRLVKGAKGIRTLLFALMMSLP

ALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDM

FNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDC

DPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVV

NMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEK

FDPDATQFIEFSKLSDFAAALDPPLLIAKPNKVQLIA

MDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLR

SQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVI

QRAYRRYRLRQNVKNISSIYIKDGDRDDDLLNKKD

MAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKE

KYEQDRTEKEDKGKDSKESKK

14
huNav157 chimera 5
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGN

VSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQATIAET

EEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLE

MSPLAPVNSHERRSKRRKRMSSGTEECGEDRLPKS

DSEDGPRAMNHLSLTRGLSRTSMKPRSSRGSIFTFR

RRDLGSEADFADDENSTAGESESHHTSLLVPWPLR

RTSAQGQPSPGTSAPGHALHGKKNSTVDCNGVVSL

LGAGDPEATSPGSHLLRPVMLEHPPDTTTPSEEPGG

PQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALE

ELEESRHKCPPCWNRLAQRYLIWECCPLWMSIKQG

VKLVVMDPFTDLTITMCIVLNTLFMALEHYNMTSE

FEEMLQVGNLVFTGIFTAEMTFKIIALDPYYYFQQG

WNIFDSIIVILSLMELGLSRMSNLSVLRSFRLLRVFK

LAKSWPTLNTLIKIIGNSVGALGNLTLVLAIIVFIFA

VVGMQLFGKNYSELRDSDSGLLPRWHMMDFFHAF

LIIFRILCGEWIETMWDCMEVSGQSLCLLVFLLVMV

IGNLVVLNLFLALLLSSFSADNLTAPDEDREMNNL

QLALARIQRGLRFVKRTTWDFCCGLLRQRPQKPAA

LAAQGQLPSCIATPYSPPPPETEKVPPTRKETRFEEG

EQPGQGTPGDPEPVCVPIAVAESDTDDQEEDEENSL

GTEEESSKQESQPVSGGPEAPPDSRTWSQVSATASS

EAEASASQADWRQQWKAEPQAPGCGETPEDSCSE

GSTADMTNTAELLEQIPDLGQDVKDPEDCFTEGCV

RRCPCCAVDTTQAPGKVWWRLRKTCYHIVEHSWF

ETFIIFMILLSSGALAFEDIYLEERKTIKVLLEYADK

MFTYVFVLEMLLKWVAYGFKKYFTNAWCWLDFL

IVDVSLVSLVANTLGFAEMGPIKSLRTLRALRPLRA

LSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSI

MGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCE

SLNLTGELYWTKVKVNFDNVGAGYLALLQVATFK

GWMDIMYAAVDSRGYEEQPQWEYNLYMYIYFVIF

IIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQ

KKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVT

NQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVL

YWINVVFIILFTGECVLKLISLRHYYFTVGWNIFDFV

VVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRL

VKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYA

IFGMSNFAYVKKEDGINDMFNFETFGNSMICLFQIT

TSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDC

GNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVATE

ESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSD

FAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCL

DILFAFTKRVLGESGEMDSLRSQMEERFMSANPSK

VSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNV

KNISSIYIKDGDRDDDLLNKKDMAFDNVNENSSPE

KTDATSSTTSPPSYDSVTKPDKEKYEQDRTEKEDK

GKDSKESKK

15
huNav157 chimera 6
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGN

VSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQATIAET

EEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLE

MSPLAPVNSHERRSKRRKRMSSGTEECGEDRLPKS

DSEDGPRAMNHLSLTRGLSRTSMKPRSSRGSIFTFR

RRDLGSEADFADDENSTAGESESHHTSLLVPWPLR

RTSAQGQPSPGTSAPGHALHGKKNSTVDCNGVVSL

LGAGDPEATSPGSHLLRPVMLEHPPDTTTPSEEPGG

PQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALE

ELEESRHKCPPCWNRLAQRYLIWECCPLWMSIKQG

VKLVVMDPFTDLTITMCIVLNTLFMALEHYNMTSE

FEEMLQVGNLVFTGIFTAEMTFKIIALDPYYYFQQG

WNIFDSIIVILSLMELGLSRMSNLSVLRSFRLLRVFK

LAKSWPTLNTLIKIIGNSVGALGNLTLVLAIIVFIFA

VVGMQLFGKNYSELRDSDSGLLPRWHMMDFFHAF

LIIFRILCGEWIETMWDCMEVSGQSLCLLVFLLVMV

IGNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNLQI

AVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQA

EDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGF

GSSVDKHLMEDSDGQSFIHNPSLTVTVPIAPGESDL

ENMNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPL

PGEGEEAEAEPMNSDEPEACFTDGCVRRFSCCQVN

IESGKGKIWWNIRKTCYKIVEHSWFESFIVLMILLSS

GALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLK

WIAYGYKTYFTNAWCWLDFLIVDVSLVTLVANTL

GYSDLGPIKSLRTLRALRPLRALSRFEGMRVVVNA

LIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYECI

NTTDGSRFPASQVPNRSECFALMNVSQNVRWKNL

KVNFDNVGLGYLSLLQVATFKGWTIIMYAAVDSV

NVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVII

DNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGS

KKPQKPIPRPLNKYQGFIFDIVTKQAFDVTIMFLICL

NMVTMMVETDDQSPEKINILAKINLLEVAIFTGECI

VKLAALRHYYFTNSWNIFDFVVVILSIVGTVLSDIIQ

KYFFSPTLFRVIRLARIGRILRLIRGAKGIRTLLFALM

MSLPALFNIGLLLFLVMFIYSIFGMANFAYVKWEA

GIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILN

TGPPYCDPTLPNSNGSRGDCGSPAVGILFFTTYIIISF

LIVVNMYIAIILENFSVATEESTEPLSEDDFDMFYEI

WEKFDPEATQFIEYSVLSDFADALSEPLRIAKPNQIS

LINMDLPMVSGDRIHCMDILFAFTKRVLGESGEMD

ALKIQMEEKFMAANPSKISYEPITTTLRRKHEEVSA

MVIQRAFRRHLLQRSLKHASFLFRQQAGSGLSEED

APEREGLIAYVMSENFSRPLGPPSSSSISSTSFPPSYD

SVTRATSDNLQVRGSDYSHSEDLADFPPSPDRDRES

IV

16
huNav157 chimera 7
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGN

VSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIR

RSRIMGLSESSSETSKLSSKSAKERRNRRKKKNQKK

LSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEGHRR

AHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGR

GRDIGSETEFADDEHSIFGDNESRRGSLFVPHRPQE

RRSSNISQASRSPPMLPVNGKMHSAVDCNGVVSLV

DGRSALMLPNGQLLPEGTTNQIHKKRRCSSYLLSE

DMLNDPNLRQRAMSRASILTNTVEELEESRQKCPP

WWYRFAHKFLIWNCSPYWIKFKKCIYFIVMDPFVD

LAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNLVF

TGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLS

LVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNM

LIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKS

YKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCG

EWIETMWDCMEVAGQAMCLIVYMMVMVIGNLVV

LNLFLALLLSSFSADNLTAPDEDREMNNLQLALARI

QRGLRFVKRTTWDFCCGLLRQRPQKPAALAAQGQ

LPSCIATPYSPPPPETEKVPPTRKETRFEEGEQPGQG

TPGDPEPVCVPIAVAESDTDDQEEDEENSLGTEEES

SKQESQPVSGGPEAPPDSRTWSQVSATASSEAEASA

SQADWRQQWKAEPQAPGCGETPEDSCSEGSTADM

TNTAELLEQIPDLGQDVKDPEDCFTEGCVRRCPCC

AVDTTQAPGKVWWRLRKTCYHIVEHSWFETFIIFM

ILLSSGALAFEDIYLEERKTIKVLLEYADKMFTYVF

VLEMLLKWVAYGFKKYFTNAWCWLDFLIVDVSL

VSLVANTLGFAEMGPIKSLRTLRALRPLRALSRFEG

MRVVVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNL

FAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTG

ELYWTKVKVNFDNVGAGYLALLQVATFKGWMDI

MYAAVDSRGYEEQPQWEYNLYMYIYFVIFIIFGSFF

TLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYN

AMKKLGSKKPQKPIPRPLNKYQGFIFDIVTKQAFDV

TIMFLICLNMVTMMVETDDQSPEKINILAKINLLFV

AIFTGECIVKLAALRHYYFTNSWNIFDFVVVILSIVG

TVLSDIIQKYFFSPTLFRVIRLARIGRILRLIRGAKGIR

TLLFALMMSLPALFNIGLLLFLVMFIYSIFGMANFA

YVKWEAGIDDMFNFQTFANSMLCLFQITTSAGWD

GLLSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILF

FTTYIIISFLIVVNMYIAIILENFSVATEESTEPLSEDD

FDMFYEIWEKFDPEATQFIEYSVLSDFADALSEPLRI

AKPNQISLINMDLPMVSGDRIHCMDILFAFTKRVLG

ESGEMDALKIQMEEKFMAANPSKISYEPITTTLRRK

HEEVSAMVIQRAFRRHLLQRSLKHASFLFRQQAGS

GLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSISSTS

FPPSYDSVTRATSDNLQVRGSDYSHSEDLADFPPSP

DRDRESIV

17
huNav157 chimera 8
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQATIAETEEKEKRFQEAMEMLK

KEHEALTIRGVDTVSRSSLEMSPLAPVNSHERRSKR

RKRMSSGTEECGEDRLPKSDSEDGPRAMNHLSLTR

GLSRTSMKPRSSRGSIFTFRRRDLGSEADFADDENS

TAGESESHHTSLLVPWPLRRTSAQGQPSPGTSAPGH

ALHGKKNSTVDCNGVVSLLGAGDPEATSPGSHLLR

PVMLEHPPDTTTPSEEPGGPQMLTSQAPCVDGFEEP

GARQRALSAVSVLTSALEELEESRHKCPPCWNRLA

QRYLIWECCPLWMSIKQGVKLVVMDPFTDLTITMC

IVLNTLFMALEHYNMTSEFEEMLQVGNLVFTGIFT

AEMTFKIIALDPYYYFQQGWNIFDSIIVILSLMELGL

SRMSNLSVLRSFRLLRVFKLAKSWPTLNTLIKIIGNS

VGALGNLTLVLAIIVFIFAVVGMQLFGKNYSELRDS

DSGLLPRWHMMDFFHAFLIIFRILCGEWIETMWDC

MEVSGQSLCLLVFLLVMVIGNLVVLNLFLALLLSSF

SADNLTAPDEDREMNNLQLALARIQRGLRFVKRTT

WDFCCGLLRQRPQKPAALAAQGQLPSCIATPYSPPP

PETEKVPPTRKETRFEEGEQPGQGTPGDPEPVCVPI

AVAESDTDDQEEDEENSLGTEEESSKQESQPVSGGP

EAPPDSRTWSQVSATASSEAEASASQADWRQQWK

AEPQAPGCGETPEDSCSEGSTADMINTAELLEQIPD

LGQDVKDPEDCFTEGCVRRCPCCAVDTTQAPGKV

WWRLRKTCYHIVEHSWFETFIIFMILLSSGALAFEDI

YLEERKTIKVLLEYADKMFTYVFVLEMLLKWVAY

GFKKYFTNAWCWLDFLIVDVSLVSLVANTLGFAE

MGPIKSLRTLRALRPLRALSRFEGMRVVVNALVGA

IPSIMNVLLVCLIFWLIFSIMGVNLFAGKFGRCINQT

EGDLPLNYTIVNNKSQCESLNLTGELYWTKVKVNF

DNVGAGYLALLQVATFKGWMDIMYAAVDSRGYE

EQPQWEYNLYMYIYFVIFIIFGSFFTLNLFIGVIIDNF

NQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKP

QKPIPRPLNKYQGFIFDIVTKQAFDVTIMFLICLNMV

TMMVETDDQSPEKINILAKINLLFVAIFTGECIVKLA

ALRHYYFTNSWNIFDFVVVILSIVGTVLSDIIQKYFF

SPTLFRVIRLARIGRILRLIRGAKGIRTLLFALMMSLP

ALFNIGLLLFLVMFIYSIFGMANFAYVKWEAGIDD

MFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPP

YCDPTLPNSNGSRGDCGSPAVGILFFTTYIIISFLIVV

NMYIAIILENFSVATEESTEPLSEDDFDMFYEIWEKF

DPEATQFIEYSVLSDFADALSEPLRIAKPNQISLINM

DLPMVSGDRIHCMDILFAFTKRVLGESGEMDALKI

QMEEKFMAANPSKISYEPITTTLRRKHEEVSAMVIQ

RAFRRHLLQRSLKHASFLFRQQAGSGLSEEDAPERE

GLIAYVMSENFSRPLGPPSSSSISSTSFPPSYDSVTRA

TSDNLQVRGSDYSHSEDLADFPPSPDRDRESIV

18
huNav157 chimera 12
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMVLTV

FCLSVFALIGLQLFMGNLRHKCVRNFTALNGTNGS

VEADGLVWESLDLYLSDPENYLLKNGTSDVLLCG

NSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAF

LALFRLMTQDCWERLYQQTLRSAGKIYMIFFMLVI

FLGSFYLVNLILAVVAMAYEEQNQANIEEAKQKEL

EFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIM

GLSESSSETSKLSSKSAKERRNRRKKKNQKKLSSGE

EKGDAEKLSKSESEDSIRRKSFHLGVEGHRRAHEK

RLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIG

SETEFADDEHSIFGDNESRRGSLFVPHRPQERRSSNI

SQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSA

LMLPNGQLLPEGTTNQIHKKRRCSSYLLSEDMLND

PNLRQRAMSRASILTNTVEELEESRQKCPPWWYRF

AHKFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICI

VLNTLFMAMEHHPMTEEFKNVLAIGNLVFTGIFAA

EMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELFL

ADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGN

SVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVC

KINDDCTLPRWHMNDFFHSFLIVFRVLCGEWIETM

WDCMEVAGQAMCLIVYMMVMVIGNLVVLNLFLA

LLLSSFSSDNLTAIEEDPDANNLQIAVTRIKKGINYV

KQTLREFILKAFSKKPKISREIRQAEDLNTKKENYIS

NHTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDS

DGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDS

DSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAEPM

NSDEPEACFTDGCVRRFSCCQVNIESGKGKIWWNI

RKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIER

KKTIKIILEYADKIFTYIFILEMLLKWIAYGYKTYFT

NAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLR

TLRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLL

VCLIFWLIFSIMGVNLFAGKFYECINTTDGSRFPASQ

VPNRSECFALMNVSQNVRWKNLKVNFDNVGLGY

LSLLQVATFKGWTIIMYAAVDSVNVDKQPKYEYSL

YMYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQKKKLG

GQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNK

IQGCIFDLVTNQAFDISIMVLICLNMVTMMVEKEGQ

SQHMTEVLYWINVVFIILFTGECVLKLISLRHYYFT

VGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVI

RLARIGRILRLVKGAKGIRTLLFALMMSLPALFNIG

LLLFLVMFIYAIFGMSNFAYVKKEDGINDMFNFETF

GNSMICLFQITTSAGWDGLLAPILNSKPPDCDPKKV

HPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNMYIA

VILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDA

TQFIEFSKLSDFAAALDPPLLIAKPNKVQLIAMDLP

MVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQME

ERFMSANPSKVSYEPITTTLKRKQEDVSATVIQRAY

RRYRLRQNVKNISSIYIKDGDRDDDLLNKKDMAFD

NVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQ

DRTEKEDKGKDSKESKK

19
huNav157 chimera 14
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFSMLIMCTILTNC

IFMTMNNPPDWTKNVEYTFTGIYTFESLVKILARGF

CLHAFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVS

ALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLADV

MILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNE

TLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTD

SGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALF

RLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLG

SFYLINLILAVVAMAYEEQNQATIAETEEKEKRFQE

AMEMLKKEHEALTIRGVDTVSRSSLEMSPLAPVNS

HERRSKRRKRMSSGTEECGEDRLPKSDSEDGPRAM

NHLSLTRGLSRTSMKPRSSRGSIFTFRRRDLGSEAD

FADDENSTAGESESHHTSLLVPWPLRRTSAQGQPSP

GTSAPGHALHGKKNSTVDCNGVVSLLGAGDPEAT

SPGSHLLRPVMLEHPPDTTTPSEEPGGPQMLTSQAP

CVDGFEEPGARQRALSAVSVLTSALEELEESRHKCP

PCWNRLAQRYLIWECCPLWMSIKQGVKFIVMDPF

VDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNL

VFTGIFAAEMVLKLIAMDPYYYFQQGWNIFDSLIVT

LSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLN

TLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGK

SYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLC

GEWIETMWDCMEVAGQAMCLIVYMMVMVIGNLV

VLNLFLALLLSSFSADNLTAPDEDREMNNLQLALA

RIQRGLRFVKRTTWDFCCGLLRQRPQKPAALAAQG

QLPSCIATPYSPPPPETEKVPPTRKETRFEEGEQPGQ

GTPGDPEPVCVPIAVAESDTDDQEEDEENSLGTEEE

SSKQESQPVSGGPEAPPDSRTWSQVSATASSEAEAS

ASQADWRQQWKAEPQAPGCGETPEDSCSEGSTAD

MTNTAELLEQIPDLGQDVKDPEDCFTEGCVRRCPC

CAVDTTQAPGKVWWRLRKTCYHIVEHSWFESFIVL

MILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFIL

EMLLKWIAYGYKKYFTNAWCWLDFLIVDVSLVTL

VANTLGYSDLGPIKSLRTLRALRPLRALSRFEGMRV

VVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAG

KFYECINTTDGSRFPASQVPNRSECFALMNVSQNV

RWKNLKVNFDNVGLGYLSLLQVATFKGWTIIMYA

AVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLN

LFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAM

KKLGSKKPQKPIPRPLNKYQGFIFDLVTNQAFDISIM

VLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIIL

FTGECVLKLISLRHYYFTVGWNIFDFVVVIISIVGMF

LADLIETYFVSPTLFRVIRLARIGRILRLVKGAKGIR

TLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFA

YVKKEDGINDMFNFETFGNSMICLFQITTSAGWDG

LLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIF

YFVSYIIISFLVVVNMYIAVILENFSVATEESTEPLSE

DDFDMFYEIWEKFDPEATQFIEYSVLSDFADALSEP

LRIAKPNQISLINMDLPMVSGDRIHCMDILFAFTKR

VLGESGEMDALKIQMEEKFMAANPSKISYEPITTTL

RRKHEEVSAMVIQRAFRRHLLQRSLKHASFLFRQQ

AGSGLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSI

SSTSFPPSYDSVTRATSDNLQVRGSDYSHSEDLADF

PPSPDRDRESIV

20
huNav157 chimera 14-
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

beta1-beta2-beta3 viral
STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

P2A sequences italics;
PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

beta1-beta2-beta3 are in
NALYVLSPFHPIRRAAVKILVHSLFSMLIMCTILTNC

bold
IFMTMNNPPDWTKNVEYTFTGIYTFESLVKILARGF

CLHAFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVS

ALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLADV

MILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNE

TLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTD

SGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALF

RLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLG

SFYLINLILAVVAMAYEEQNQATIAETEEKEKRFQE

AMEMLKKEHEALTIRGVDTVSRSSLEMSPLAPVNS

HERRSKRRKRMSSGTEECGEDRLPKSDSEDGPRAM

NHLSLTRGLSRTSMKPRSSRGSIFTFRRRDLGSEAD

FADDENSTAGESESHHTSLLVPWPLRRTSAQGQPSP

GTSAPGHALHGKKNSTVDCNGVVSLLGAGDPEAT

SPGSHLLRPVMLEHPPDTTTPSEEPGGPQMLTSQAP

CVDGFEEPGARQRALSAVSVLTSALEELEESRHKCP

PCWNRLAQRYLIWECCPLWMSIKQGVKFIVMDPF

VDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNL

VFTGIFAAEMVLKLIAMDPYYYFQQGWNIFDSLIVT

LSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLN

TLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGK

SYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLC

GEWIETMWDCMEVAGQAMCLIVYMMVMVIGNLV

VLNLFLALLLSSFSADNLTAPDEDREMNNLQLALA

RIQRGLRFVKRTTWDFCCGLLRQRPQKPAALAAQG

QLPSCIATPYSPPPPETEKVPPTRKETRFEEGEQPGQ

GTPGDPEPVCVPIAVAESDTDDQEEDEENSLGTEEE

SSKQESQPVSGGPEAPPDSRTWSQVSATASSEAEAS

ASQADWRQQWKAEPQAPGCGETPEDSCSEGSTAD

MTNTAELLEQIPDLGQDVKDPEDCFTEGCVRRCPC

CAVDTTQAPGKVWWRLRKTCYHIVEHSWFESFIVL

MILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFIL

EMLLKWIAYGYKKYFTNAWCWLDFLIVDVSLVTL

VANTLGYSDLGPIKSLRTLRALRPLRALSRFEGMRV

VVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAG

KFYECINTTDGSRFPASQVPNRSECFALMNVSQNV

RWKNLKVNFDNVGLGYLSLLQVATFKGWTIIMYA

AVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLN

LFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAM

KKLGSKKPQKPIPRPLNKYQGFIFDLVTNQAFDISIM

VLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIIL

FTGECVLKLISLRHYYFTVGWNIFDFVVVIISIVGMF

LADLIETYFVSPTLFRVIRLARIGRILRLVKGAKGIR

TLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFA

YVKKEDGINDMFNFETFGNSMICLFQITTSAGWDG

LLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIF

YFVSYIIISFLVVVNMYIAVILENFSVATEESTEPLSE

DDFDMFYEIWEKFDPEATQFIEYSVLSDFADALSEP

LRIAKPNQISLINMDLPMVSGDRIHCMDILFAFTKR

VLGESGEMDALKIQMEEKFMAANPSKISYEPITTTL

RRKHEEVSAMVIQRAFRRHLLQRSLKHASFLFRQQ

AGSGLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSI

SSTSFPPSYDSVTRATSDNLQVRGSDYSHSEDLADF

PPSPDRDRESIVSGRGSGATNFSLLKQAGDVEENPGP

MGRLLALVVGAALVSSACGGCVEVDSETEAVY

GMTFKILCISCKRRSETNAETFTEWTFRQKGTE

EFVKILRYENEVLQLEEDERFEGRVVWNGSRGT

KDLQDLSIFITNVTYNHSGDYECHVYRLLFFENY

EHNTSVVKKIHIEVVDKANRDMASIVSEIMMYV

LIVVLTIWLVAEMIYCYKKIAAATETAAQENASE

YLAITSESKENCTGVQVAE
GSGATNFSLLKQAGDV

EENPGP
MHRDAWLPRPAFSLTGLSLFFSLVPPGR

SMEVTVPATLNVLNGSDARLPCTFNSCYTVNHK

QFSLNWTYQECNNCSEEMFLQFRMKIINLKLER

FQDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNC

YIMNPPDRHRGHGKIHLQVLMEEPPERDSTVAV

IVGASVGGFLAVVILVLMVVKCVRRKKEQKLST

DDLKTEEEGKTDGEGNPDDGAK
GSGATNFSLLKQ

AGDVEENPGP
MPAFNRLFPLASLVLIYWVSVCFP

VCVEVPSETEAVQGNPMKLRCISCMKREEVEAT

TVVEWFYRPEGGKDFLIYEYRNGHQEVESPFQG

RLQWNGSKDLQDVSITVLNVTLNDSGLYTCNVS

REFEFEAHRPFVKTTRLIPLRVTEEAGEDFTSVV

SEIMMYILLVFLTLWLLIEMIYCYRKVSKAEEAA

QENASDYLAIPSENKENSAVPVEE

21
beta1-beta2-beta3 viral

MGRLLALVVGAALVSSACGGCVEVDSETEAVY

P2A sequences italics;

GMTFKILCISCKRRSETNAETFTEWTFRQKGTE

beta1-beta2-beta3 are in

EFVKILRYENEVLQLEEDERFEGRVVWNGSRGT

bold

KDLQDLSIFITNVTYNHSGDYECHVYRLLFFENY

EHNTSVVKKIHIEVVDKANRDMASIVSEIMMYV

LIVVLTIWLVAEMIYCYKKIAAATETAAQENASE

YLAITSESKENCTGVQVAE
GSGATNFSLLKQAGDV

EENPGP
MHRDAWLPRPAFSLTGLSLFFSLVPPGR

SMEVTVPATLNVLNGSDARLPCTFNSCYTVNHK

QFSLNWTYQECNNCSEEMFLQFRMKIINLKLER

FQDRVEFSGNPSKYDVSVMLRNVQPEDEGIYNC

YIMNPPDRHRGHGKIHLQVLMEEPPERDSTVAV

IVGASVGGFLAVVILVLMVVKCVRRKKEQKLST

DDLKTEEEGKTDGEGNPDDGAK
GSGATNFSLLKQ

AGDVEENPGP
MPAFNRLFPLASLVLIYWVSVCFP

VCVEVPSETEAVQGNPMKLRCISCMKREEVEAT

TVVEWFYRPEGGKDFLIYEYRNGHQEVESPFQG

RLQWNGSKDLQDVSITVLNVTLNDSGLYTCNVS

REFEFEAHRPFVKTTRLIPLRVTEEAGEDFTSVV

SEIMMYILLVFLTLWLLIEMIYCYRKVSKAEEAA

QENASDYLAIPSENKENSAVPVEE

22
huNav1.5-beta1-beta2-
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

beta3 viral P2A
STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

sequences italics; beta1-
PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

beta2-beta3 are in bold
NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYTTEFVDLGN

VSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQATIAET

EEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLE

MSPLAPVNSHERRSKRRKRMSSGTEECGEDRLPKS

DSEDGPRAMNHLSLTRGLSRTSMKPRSSRGSIFTFR

RRDLGSEADFADDENSTAGESESHHTSLLVPWPLR

RTSAQGQPSPGTSAPGHALHGKKNSTVDCNGVVSL

LGAGDPEATSPGSHLLRPVMLEHPPDTTTPSEEPGG

PQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALE

ELEESRHKCPPCWNRLAQRYLIWECCPLWMSIKQG

VKLVVMDPFTDLTITMCIVLNTLFMALEHYNMTSE

FEEMLQVGNLVFTGIFTAEMTFKIIALDPYYYFQQG

WNIFDSIIVILSLMELGLSRMSNLSVLRSFRLLRVFK

LAKSWPTLNTLIKIIGNSVGALGNLTLVLAIIVFIFA

VVGMQLFGKNYSELRDSDSGLLPRWHMMDFFHAF

LIIFRILCGEWIETMWDCMEVSGQSLCLLVFLLVMV

IGNLVVLNLFLALLLSSFSADNLTAPDEDREMNNL

QLALARIQRGLRFVKRTTWDFCCGLLRQRPQKPAA

LAAQGQLPSCIATPYSPPPPETEKVPPTRKETRFEEG

EQPGQGTPGDPEPVCVPIAVAESDTDDQEEDEENSL

GTEEESSKQESQPVSGGPEAPPDSRTWSQVSATASS

EAEASASQADWRQQWKAEPQAPGCGETPEDSCSE

GSTADMTNTAELLEQIPDLGQDVKDPEDCFTEGCV

RRCPCCAVDTTQAPGKVWWRLRKTCYHIVEHSWF

ETFIIFMILLSSGALAFEDIYLEERKTIKVLLEYADK

MFTYVFVLEMLLKWVAYGFKKYFTNAWCWLDFL

IVDVSLVSLVANTLGFAEMGPIKSLRTLRALRPLRA

LSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSI

MGVNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCE

SLNLTGELYWTKVKVNFDNVGAGYLALLQVATFK

GWMDIMYAAVDSRGYEEQPQWEYNLYMYIYFVIF

IIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQ

KKYYNAMKKLGSKKPQKPIPRPLNKYQGFIFDIVT

KQAFDVTIMFLICLNMVTMMVETDDQSPEKINILA

KINLLEVAIFTGECIVKLAALRHYYFTNSWNIFDFV

VVILSIVGTVLSDIIQKYFFSPTLFRVIRLARIGRILRL

IRGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSI

FGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQI

TTSAGWDGLLSPILNTGPPYCDPTLPNSNGSRGDCG

SPAVGILFFTTYIIISFLIVVNMYIAIILENFSVATEEST

EPLSEDDFDMFYEIWEKFDPEATQFIEYSVLSDFAD

ALSEPLRIAKPNQISLINMDLPMVSGDRIHCMDILFA

FTKRVLGESGEMDALKIQMEEKFMAANPSKISYEPI

TTTLRRKHEEVSAMVIQRAFRRHLLQRSLKHASFLF

RQQAGSGLSEEDAPEREGLIAYVMSENFSRPLGPPS

SSSISSTSFPPSYDSVTRATSDNLQVRGSDYSHSEDL

ADFPPSPDRDRESIVSGRGSGATNFSLLKQAGDVEEN

PGP
MGRLLALVVGAALVSSACGGCVEVDSETEA

VYGMTFKILCISCKRRSETNAETFTEWTFRQKG

TEEFVKILRYENEVLQLEEDERFEGRVVWNGSR

GTKDLQDLSIFITNVTYNHSGDYECHVYRLLFFE

NYEHNTSVVKKIHIEVVDKANRDMASIVSEIMM

YVLIVVLTIWLVAEMIYCYKKIAAATETAAQEN

ASEYLAITSESKENCTGVQVAE
GSGATNFSLLKQA

GDVEENPGP
MHRDAWLPRPAFSLTGLSLFFSLVP

PGRSMEVTVPATLNVLNGSDARLPCTFNSCYTV

NHKQFSLNWTYQECNNCSEEMFLQFRMKIINLK

LERFQDRVEFSGNPSKYDVSVMLRNVQPEDEGI

YNCYIMNPPDRHRGHGKIHLQVLMEEPPERDST

VAVIVGASVGGFLAVVILVLMVVKCVRRKKEQK

LSTDDLKTEEEGKTDGEGNPDDGAK
GSGATNFSL

LKQAGDVEENPGPMPAFNRLFPLASLVLIYWVSV

CFPVCVEVPSETEAVQGNPMKLRCISCMKREEV

EATTVVEWFYRPEGGKDFLIYEYRNGHQEVESP

FQGRLQWNGSKDLQDVSITVLNVTLNDSGLYTC

NVSREFEFEAHRPFVKTTRLIPLRVTEEAGEDFT

SVVSEIMMYILLVFLTLWLLIEMIYCYRKVSKAE

EAAQENASDYLAIPSENKENSAVPVEE

23
huNav1.1 (alpha
MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKN

subunit)
PKPDKKDDDENGPKPNSDLEAGKNLPFIYGDIPPEM

VSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALY

ILTPFNPLRKIAIKILVHSLFSMLIMCTILTNCVFMTM

SNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFT

FLRDPWNWLDFTVITFAYVTEFVDLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSI

EKNITVNYNGTLINETVFEFDWKSYIQDSRYHYFLE

GFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGY

TSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGK

TYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQAT

LEEAEQKEAEFQQMIEQLKKQQEAAQQAATATAS

EHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKK

RKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIE

GNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRTSLFS

FRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVP

RRHGERRNSNLSQTSRSSRMLAVFPANGKMHSTV

DCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDN

GTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSI

ASILTNTVEELEESRQKCPPCWYKFSNIFLIWDCSPY

WLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAM

EHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAM

DPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVL

RSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLT

LVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLP

RWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAG

QAMCLTVFMMVMVIGNLVVLNLFLALLLSSFSAD

NLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIY

EFIQQSFIRKQKILDEIKPLDDLNNKKDSCMSNHTA

EIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYM

SFINNPSLTVTVPIAVGESDFENLNTEDFSSESDLEES

KEKLNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPE

ACFTEGCVQRFKCCQINVEEGRGKQWWNLRRTCF

RIVEHNWFETFIVFMILLSSGALAFEDIYIDQRKTIK

TMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNA

WCWLDFLIVDVSLVSLTANALGYSELGAIKSLRTL

RALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLV

CLIFWLIFSIMGVNLFAGKFYHCINTTTGDRFDIEDV

NNHTDCLKLIERNETARWKNVKVNFDNVGFGYLS

LLQVATFKGWMDIMYAAVDSRNVELQPKYEESLY

MYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQ

DIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQ

GMVFDFVTRQVFDISIMILICLNMVTMMVETDDQS

EYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIG

WNIFDFVVVILSIVGMFLAELIEKYFVSPTLFRVIRL

ARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLL

FLVMFIYAIFGMSNFAYVKREVGIDDMFNFETFGNS

MICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPG

SSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIAVILE

NFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQF

MEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMV

SGDRIHCLDILFAFTKRVLGESGEMDALRIQMEERF

MASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRH

LLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRIN

ENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEG

KDEKAKGK

24
huNav1.2 (alpha
MAQSVLVPPGPDSFRFFTRESLAAIEQRIAEEKAKR

subunit)
PKQERKDEDDENGPKPNSDLEAGKSLPFIYGDIPPE

MVSVPLEDLDPYYINKKTFIVLNKGKAISRFSATPA

LYILTPFNPIRKLAIKILVHSLFNMLIMCTILTNCVFM

TMSNPPDWTKNVEYTFTGIYTFESLIKILARGFCLE

DFTFLRDPWNWLDFTVITFAYVTEFVDLGNVSALR

TFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMIL

TVFCLSVFALIGLQLFMGNLRNKCLQWPPDNSSFEI

NITSFFNNSLDGNGTTFNRTVSIFNWDEYIEDKSHF

YFLEGQNDALLCGNSSDAGQCPEGYICVKAGRNPN

YGYTSFDTFSWAFLSLFRLMTQDFWENLYQLTLRA

AGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQN

QATLEEAEQKEAEFQQMLEQLKKQQEEAQAAAAA

ASAESRDFSGAGGIGVFSESSSVASKLSSKSEKELK

NRRKKKKQKEQSGEEEKNDRVRKSESEDSIRRKGF

RFSLEGSRLTYEKRFSSPHQSLLSIRGSLFSPRRNSR

ASLFSFRGRAKDIGSENDFADDEHSTFEDNDSRRDS

LFVPHRHGERRHSNVSQASRASRVLPILPMNGKMH

SAVDCNGVVSLVGGPSTLTSAGQLLPEGTTTETEIR

KRRSSSYHVSMDLLEDPTSRQRAMSIASILTNTMEE

LEESRQKCPPCWYKFANMCLIWDCCKPWLKVKHL

VNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTEQ

FSSVLSVGNLVFTGIFTAEMFLKIIAMDPYYYFQEG

WNIFDGFIVSLSLMELGLANVEGLSVLRSFRLLRVF

KLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIF

AVVGMQLFGKSYKECVCKISNDCELPRWHMHDFF

HSFLIVFRVLCGEWIETMWDCMEVAGQTMCLTVF

MMVMVIGNLVVLNLFLALLLSSFSSDNLAATDDD

NEMNNLQIAVGRMQKGIDFVKRKIREFIQKAFVRK

QKALDEIKPLEDLNNKKDSCISNHTTIEIGKDLNYL

KDGNGTTSGIGSSVEKYVVDESDYMSFINNPSLTVT

VPIAVGESDFENLNTEEFSSESDMEESKEKLNATSSS

EGSTVDIGAPAEGEQPEVEPEESLEPEACFTEDCVR

KFKCCQISIEEGKGKLWWNLRKTCYKIVEHNWFET

FIVFMILLSSGALAFEDIYIEQRKTIKTMLEYADKVF

TYIFILEMLLKWVAYGFQVYFTNAWCWLDFLIVDV

SLVSLTANALGYSELGAIKSLRTLRALRPLRALSRF

EGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYHCINYTTGEMFDVSVVNNYSECKALIE

SNQTARWKNVKVNFDNVGLGYLSLLQVATFKGW

MDIMYAAVDSRNVELQPKYEDNLYMYLYFVIFIIF

GSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQK

KYYNAMKKLGSKKPQKPIPRPANKFQGMVFDFVT

KQVFDISIMILICLNMVTMMVETDDQSQEMTNILY

WINLVFIVLFTGECVLKLISLRYYYFTIGWNIFDFVV

VILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLI

KGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIF

GMSNFAYVKREVGIDDMFNFETFGNSMICLFQITTS

AGWDGLLAPILNSGPPDCDPDKDHPGSSVKGDCGN

PSVGIFFFVSYIIISFLVVVNMYIAVILENFSVATEES

AEPLSEDDFEMFYEVWEKFDPDATQFIEFAKLSDFA

DALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDIL

FAFTKRVLGESGEMDALRIQMEERFMASNPSKVSY

EPITTTLKRKQEEVSAIIIQRAYRRYLLKQKVKKVSS

IYKKDKGKECDGTPIKEDTLIDKLNENSTPEKTDMT

PSTTSPPSYDSVTKPEKEKFEKDKSEKEDKGKDIRE

SKK

25
huNav1.3 (alpha
MAQALLVPPGPESFRLFTRESLAAIEKRAAEEKAKK

subunit)
PKKEQDNDDENKPKPNSDLEAGKNLPFIYGDIPPE

MVSEPLEDLDPYYINKKTFIVMNKGKAIFRFSATSA

LYILTPLNPVRKIAIKILVHSLFSMLIMCTILTNCVFM

TLSNPPDWTKNVEYTFTGIYTFESLIKILARGFCLED

FTFLRDPWNWLDFSVIVMAYVTEFVSLGNVSALRT

FRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILT

VFCLSVFALIGLQLFMGNLRNKCLQWPPSDSAFET

NTTSYFNGTMDSNGTFVNVTMSTFNWKDYIGDDS

HFYVLDGQKDPLLCGNGSDAGQCPEGY

ICVKAGRNPNYGYTSFDTFSWAFLSLFRLMTQDYW

ENLYQLTLRAAGKTYMIFFVLVIFLGSFYLVNLILA

VVAMAYEEQNQATLEEAEQKEAEFQQMLEQLKK

QQEEAQAVAAASAASRDFSGIGGLGELLESSSEASK

LSSKSAKEWRNRRKKRRQREHLEGNNKGERDSFP

KSESEDSVKRSSFLFSMDGNRLTSDKKFCSPHQSLL

SIRGSLFSPRRNSKTSIFSFRGRAKDVGSENDFADDE

HSTFEDSESRRDSLFVPHRHGERRNSNVSQASMSSR

MVPGLPANGKMHSTVDCNGVVSLVGGPSALTSPT

GQLPPEGTTTETEVRKRRLSSYQISMEMLEDSSGRQ

RAVSIASILTNTMEELEESRQKCPPCWYRFANVFLI

WDCCDAWLKVKHLVNLIVMDPFVDLAITICI

VLNTLFMAMEHYPMTEQFSSVLTVGNLVFTGIFTA

EMVLKIIAMDPYYYFQEGWNIFDGIIVSLSLMELGL

SNVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGN

SVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVC

KINDDCTLPRWHMNDFFHSFLIVFRVLCGEWIETM

WDCMEVAGQTMCLIVFMLVMVIGNLVVLNLFLAL

LLSSFSSDNLAATDDDNEMNNLQIAVGRMQKGIDY

VKNKMRECFQKAFFRKPKVIEIHEGNKIDSCMSNN

TGIEISKELNYLRDGNGTTSGVGTGSSVEKYVIDEN

DYMSFINNPSLTVTVPIAVGESDFENLNTEEFSSESE

LEESKEKLNATSSSEGSTVDVVLPREGEQAETEPEE

DLKPEACFTEGCIKKFPFCQVSTEEGKGK

IWWNLRKTCYSIVEHNWFETFIVFMILLSSGALAFE

DIYIEQRKTIKTMLEYADKVFTYIFILEMLLKWVAY

GFQTYFTNAWCWLDFLIVDVSLVSLVANALGYSEL

GAIKSLRTLRALRPLRALSRFEGMRVVVNALVGAIP

SIMNVLLVCLIFWLIFSIMGVNLFAGKFYHCVNMTT

GNMFDISDVNNLSDCQALGKQARWKNVKVNFDN

VGAGYLALLQVATFKGWMDIMYAAVDSRDVKLQ

PVYEENLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQ

QKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKP

IPRPANKFQGMVFDFVTRQVFDISIMILICLNMVTM

MVETDDQGKYMTLVLSRINLVFIVLFTGEFVLKLV

SLRHYYFTIGWNIFDFVVVILSIVGMFLAEMI

EKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFAL

MMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKE

AGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPIL

NSAPPDCDPDTIHPGSSVKGDCGNPSVGIFFFVSYIII

SFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMF

YEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKP

NKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGES

GEMDALRIQMEDRFMASNPSKVSYEPITTTLKRKQ

EEVSAAIIQRNFRCYLLKQRLKNISSNYNKEAIKGRI

DLPIKQDMIIDKLNGNSTPEKTDGSSSTTSPPSYDSV

TKPDKEKFEKDKPEKESKGKEVRENQK

26
huNav1.4 (alpha
MARPSLCTLVPLGPECLRPFTRESLAAIEQRAVEEE

subunit)
ARLQRNKQMEIEEPERKPRSDLEAGKNLPMIYGDP

PPEVIGIPLEDLDPYYSNKKTFIVLNKGKAIFRFSAT

PALYLLSPFSVVRRGAIKVLIHALFSMFIMITILTNC

VFMTMSDPPPWSKNVEYTFTGIYTFESLIKILARGF

CVDDFTFLRDPWNWLDFSVIMMAYLTEFVDLGNIS

ALRTFRVLRALKTITVIPGLKTIVGALIQSVKKLSDV

MILTVFCLSVFALVGLQLFMGNLRQKCVRWPPPFN

DTNTTWYSNDTWYGNDTWYGNEMWYGNDSWY

ANDTWNSHASWATNDTFDWDAYISDEGNFYFLEG

SNDALLCGNSSDAGHCPEGYECIKTGRNPNYGYTS

YDTFSWAFLALFRLMTQDYWENLFQLTLRAAGKT

YMIFFVVIIFLGSFYLINLILAVVAMAYAEQNEATL

AEDKEKEEEFQQMLEKFKKHQEELEKAKAAQALE

GGEADGDPAHGKDCNGSLDTSQGEKGAPRQSSSG

DSGISDAMEELEEAHQKCPPWWYKCAHKVLIWNC

CAPWLKFKNIIHLIVMDPFVDLGITICIVLNTLFMA

MEHYPMTEHFDNVLTVGNLVFTGIFTAEMVLKLIA

MDPYEYFQQGWNIFDSIIVTLSLVELGLANVQGLSV

LRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNL

TLVLAIIVFIFAVVGMQLFGKSYKECVCKIALDCNL

PRWHMHDFFHSFLIVFRILCGEWIETMWDCMEVA

GQAMCLTVFLMVMVIGNLVVLNLFLALLLSSFSAD

SLAASDEDGEMNNLQIAIGRIKLGIGFAKAFLLGLL

HGKILSPKDIMLSLGEADGAGEAGEAGETAPEDEK

KEPPEEDLKKDNHILNHMGLADGPPSSLELDHLNFI

NNPYLTIQVPIASEESDLEMPTEEETDTFSEPEDSKK

PPQPLYDGNSSVCSTADYKPPEEDPEEQAEENPEGE

QPEECFTEACVQRWPCLYVDISQGRGKKWWTLRR

ACFKIVEHNWFETFIVFMILLSSGALAFEDIYIEQRR

VIRTILEYADKVFTYIFIMEMLLKWVAYGFKVYFT

NAWCWLDFLIVDVSIISLVANWLGYSELGPIKSLRT

LRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLL

VCLIFWLIFSIMGVNLFAGKFYYCINTTTSERFDISE

VNNKSECESLMHTGQVRWLNVKVNYDNVGLGYL

SLLQVATFKGWMDIMYAAVDSREKEEQPQYEVNL

YMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKLG

GKDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPQNK

IQGMVYDLVTKQAFDITIMILICLNMVTMMVETDN

QSQLKVDILYNINMIFIIIFTGECVLKMLALRQYYFT

VGWNIFDFVVVILSIVGLALSDLIQKYFVSPTLFRVI

RLARIGRVLRLIRGAKGIRTLLFALMMSLPALFNIG

LLLFLVMFIYSIFGMSNFAYVKKESGIDDMFNFETF

GNSIICLFEITTSAGWDGLLNPILNSGPPDCDPNLEN

PGTSVKGDCGNPSIGICFFCSYIIISFLIVVNMYIAIIL

ENFNVATEESSEPLGEDDFEMFYETWEKFDPDATQ

FIAYSRLSDFVDTLQEPLRIAKPNKIKLITLDLPMVP

GDKIHCLDILFALTKEVLGDSGEMDALKQTMEEKF

MAANPSKVSYEPITTTLKRKHEEVCAIKIQRAYRRH

LLQRSMKQASYMYRHSHDGSGDDAPEKEGLLANT

MSKMYGHENGNSSSPSPEEKGEAGDAGPTMGLMP

ISPSDTAWPPAPPPGQTVRPGVKESLV

27
huNav1.5 (alpha
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARG

subunit)
STTLQESREGLPEEEAPRPQLDLQASKKLPDLYGNP

PQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFRFSAT

NALYVLSPFHPIRRAAVKILVHSLFNMLIMCTILTN

CVFMAQHDPPPWTKYVEYTFTAIYTFESLVKILAR

GFCLHAFTFLRDPWNWLDFSVIIMAYVSENIKLGN

LSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLA

DVMVLTVFCLSVFALIGLQLFMGNLRHKCVRNFTA

LNGTNGSVEADGLVWESLDLYLSDPENYLLKNGTS

DVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFD

SFAWAFLALFRLMTQDCWERLYQQTLRSAGKIYMI

FFMLVIFLGSFYLVNLILAVVAMAYEEQNQATIAET

EEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLE

MSPLAPVNSHERRSKRRKRMSSGTEECGEDRLPKS

DSEDGPRAMNHLSLTRGLSRTSMKPRSSRGSIFTFR

RRDLGSEADFADDENSTAGESESHRTSLLVPWPLR

RTSAQGQPSPGTSAPGHALHGKKNSTVDCNGVVSL

LGAGDPEATSPGSHLLRPVMLEHPPDTTTPSEEPGG

PQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALE

ELEESRHKCPPCWNRLAQRYLIWECCPLWMSIKQG

VKLVVMDPFTDLTITMCIVLNTLFMALEHYNMTSE

FEEMLQVGNLVFTGIFTAEMTFKIIA

LDPYYYFQQGWNIFDSIIVILSLMELGLSRMSNLSV

LRSFRLLRVFKLAKSWPTLNTLIKIIGNSVGALGNL

TLVLAIIVFIFAVVGMQLFGKNYSELRDSDSGLLPR

WHMMDFFHAFLIIFRILCGEWIETMWDCMEVSGQS

LCLLVFLLVMVIGNLVVLNLFLALLLSSFSADNLTA

PDEDREMNNLQLALARIQRGLRFVKRTTWDFCCG

LLRQRPQKPAALAAQGQLPSCIATPYSPPPPETEKV

PPTRKETRFEEGEQPGQGTPGDPEPVCVPIAVAESD

TDDQEEDEENSLGTEEESSKQESQPVSGGPEAPPDS

RTWSQVSATASSEAEASASQADWRQQWKAEPQAP

GCGETPEDSCSEGSTADMTNTAELLEQIPDLGQDV

KDPEDCFTEGCVRRCPCCAVDTTQAPGKVWWRLR

KTCYHIVEHSWFETFIIFMILLSSGALAFEDIYLEER

KTIKVLLEYADKMFTYVFVLEMLLKWVAYGFKKY

FTNAWCWLDFLIVDVSLVSLVANTLGFAEMGPIKS

LRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMN

VLLVCLIFWLIFSIMGVNLFAGKFGRCINQTEGDLP

LNYTIVNNKSQCESLNLTGELYWTKVKVNFDNVG

AGYLALLQVATFKGWMDIMYAAVDSRGYEEQPQ

WEYNLYMYIYFVIFIIFGSFFTLNLFIGVIIDNFNQQK

KKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIP

RPLNKYQGFIFDIVTKQAFDVTIMFLICLN

MVTMMVETDDQSPEKINILAKINLLFVAIFTGTVLS

DIIQKYFFSPTLFRVIRLARIGRILRLIRGAKGIRTLLF

ALMMSLPALFNIGLLLFLVMFIYSIFGMANFAYVK

WEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLS

PILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTTY

IIISFLIVVNMYIAIILENFSVATEESTEPLSEDDFDMF

YEIWEKFDPEATQFIEYSVLSDFADALSEPLRIAKPN

QISLINMDLPMVSGDRIHCMDI

LFAFTKRVLGESGEMDALKIQMEEKFMAANPSKIS

YEPITTTLRRKHEEVSAMVIQRAFRRHLLQRSLKHA

SFLFRQQAGSGLSEEDAPEREGLIAYVMSENFSRPL

GPPSSSSISSTSFPPSYDSVTRATSDNLQVRGSDYSH

SEDLADFPPSPDRDRESIV

28
huNav1.6 (alpha
MAARLLAPPGPDSFKPFTPESLANIERRIAESKLKKP

subunit)
PKADGSHREDDEDSKPKPNSDLEAGKSLPFIYGDIP

QGLVAVPLEDFDPYYLTQKTFVVLNRGKTLFRFSA

TPALYILSPFNLIRRIAIKILIHSVFSMIIMCTILTNCVF

MTFSNPPDWSKNVEYTFTGIYTFESLVKIIARGFCID

GFTFLRDPWNWLDFSVIMMAYITEFVNLGNVSALR

TFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMIL

TVFCLSVFALIGLQLFMGNLRNKCVVWPINFNESY

LENGTKGFDWEEYINNKTNFYTVPGMLEPLLCGNS

SDAGQCPEGYQCMKAGRNPNYGYTSFDTFSWAFL

ALFRLMTQDYWENLYQLTLRAAGKTYMIFFVLVIF

VGSFYLVNLILAVVAMAYEEQNQATLEEAEQKEA

EFKAMLEQLKKQQEEAQAAAMATSAGTVSEDAIE

EEGEEGGGSPRSSSEISKLSSKSAKERRNRRKKRKQ

KELSEGEEKGDPEKVFKSESEDGMRRKAFRLPDNR

IGRKFSIMNQSLLSIPGSPFLSRHNSKSSIFSFRGPGR

FRDPGSENEFADDEHSTVEESEGRRDSLFIPIRARER

RSSYSGYSGYSQGSRSSRIFPSLRRSVKRNSTVDCN

GVVSLIGGPGSHIGGRLLPEATTEVEIKKKGPGSLL

VSMDQLASYGRKDRINSIMSVVTNTLVEELEESQR

KCPPCWYKFANTFLIWECHPYWIKLKEIVNLIVMD

PFVDLAITICIVLNTLFMAMEHHPMTPQFEHVLAVG

NLVFTGIFTAEMFLKLIAMDPYYYFQEGWNIFDGFI

VSLSLMELSLADVEGLSVLRSFRLLRVFKLAKSWP

TLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQL

FGKSYKECVCKINQDCELPRWHMHDFFHSFLIVFR

VLCGEWIETMWDCMEVAGQAMCLIVFMMVMVIG

NLVVLNLFLALLLSSFSADNLAATDDDGEMNNLQI

SVIRIKKGVAWTKLKVHAFMQAHFKQREADEVKP

LDELYEKKANCIANHTGADIHRNGDFQKNGNGTTS

GIGSSVEKYIIDEDHMSFINNPNLTVRVPIAVGESDF

ENLNTEDVSSESDPEGSKDKLDDTSSSEGSTIDIKPE

VEEVPVEQPEEYLDPDACFTEGCVQRFKCCQVNIE

EGLGKSWWILRKTCFLIVEHNWFETFIIFMILLSSGA

LAFEDIYIEQRKTIRTILEYADKVFTYIFILEMLLKW

TAYGFVKFFTNAWCWLDFLIVAVSLVSLIANALGY

SELGAIKSLRTLRALRPLRALSRFEGMRVVVNALV

GAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKYHYCF

NETSEIRFEIEDVNNKTECEKLMEGNNTEIRWKNV

KINFDNVGAGYLALLQVATFKGWMDIMYAAVDSR

KPDEQPKYEDNIYMYIYFVIFIIFGSFFTLNLFIGVIID

NFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSK

KPQKPIPRPLNKIQGIVFDFVTQQAFDIVIMMLICLN

MVTMMVETDTQSKQMENILYWINLVFVIFFTCECV

LKMFALRHYYFTIGWNIFDFVVVILSIVGMFLADIIE

KYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFAL

MMSLPALFNIGLLLFLVMFIFSIFGMSNFAYVKHEA

GIDDMFNFETFGNSMICLFQITTSAGWDGLLLPILN

RPPDCSLDKEHPGSGFKGDCGNPSVGIFFFVSYI

IISFLIVVNMYIAIILENFSVATEESADPLSEDDFETF

YEIWEKFDPDATQFIEYCKLADFADALEHPLRVPKP

NTIELIAMDLPMVSGDRIHCLDILFAFTKRVLGDSG

ELDILRQQMEERFVASNPSKVSYEPITTTLRRKQEE

VSAVVLQRAYRGHLARRGFICKKTTSNKLENGGTH

REKKESTPSTASLPSYDSVTKPEKEKQQRAEEGRRE

RAKRQKEVRESKC

29
huNav1.8 (alpha
MEFPIGSLETNNFRRFTPESLVEIEKQIAAKQGTKKA

subunit)
REKHREQKDQEEKPRPQLDLKACNQLPKFYGELPA

ELIGEPLEDLDPFYSTHRTFMVLNKGRTISRFSATRA

LWLFSPFNLIRRTAIKVSVHSWFSLFITVTILVNCVC

MTRTDLPEKIEYVFTVIYTFEALIKILARGFCLNEFT

YLRDPWNWLDFSVITLAYVGTAIDLRGISGLRTFRV

LRALKTVSVIPGLKVIVGALIHSVKKLADVTILTIFC

LSVFALVGLQLFKGNLKNKCVKNDMAVNETTNYS

SHRKPDIYINKRGTSDPLLCGNGSDSGHCPDGYICL

KTSDNPDFNYTSFDSFAWAFLSLFRLMTQDSWERL

YQQTLRTSGKIYMIFFVLVIFLGSFYLVNLILAVVT

MAYEEQNQATTDEIEAKEKKFQEALEMLRKEQEV

LAALGIDTTSLHSHNGSPLTSKNASERRHRIKPRVS

EGSTEDNKSPRSDPYNQRRMSFLGLASGKRRASHG

SVFHFRSPGRDISLPEGVTDDGVFPGDHESHRGSLL

LGGGAGQQGPLPRSPLPQPSNPDSRHGEDEHQPPPT

SELAPGAVDVSAFDAGQKKTFLSAEYLDEPFRAQR

AMSVVSIITSVLEELEESEQKCPPCLTSLSQKYLIWD

CCPMWVKLKTILFGLVTDPFAELTITLCIVVNTIFM

AMEHHGMSPTFEAMLQIGNIVFTIFFTAEMVFKIIAF

DPYYYFQKKWNIFDCIIVTVSLLELGVAKKGSLSVL

RSFRLLRVFKLAKSWPTLNTLIKIIGNSVGALGNLTI

ILAIIVFVFALVGKQLLGENYRNNRKNISAPHEDWP

RWHMHDFFHSFLIVFRILCGEWIENMWACMEVGQ

KSICLILFLTVMVLGNLVVLNLFIALLLNSFSADNLT

APEDDGEVNNLQVALARIQVFGHRTKQALCSFFSR

SCPFPQPKAEPELVVKLPLSSSKAENHIAANTARGS

SGGLQAPRGPRDEHSDFIANPTVWVSVPIAEGESDL

DDLEDDGGEDAQSFQQEVIPKGQQEQLQQVERCG

DHLTPRSPGTGTSSEDLAPSLGETWKDESVPQVPAE

GVDDTSSSEGSTVDCLDPEEILRKIPELADDLEEPDD

CFTEGCIRHCPCCKLDTTKSPWDVGWQVRKTCYRI

VEHSWFESFIIFMILLSSGSLAFEDYYLDQKPTVKAL

LEYTDRVFTFIFVFEMLLKWVAYGFKKYFTNAWC

WLDFLIVNISLISLTAKILEYSEVAPIKALRTLRALRP

LRALSRFEGMRVVVDALVGAIPSIMNVLLVCLIFW

LIFSIMGVNLFAGKFWRCINYTDGEFSLVPLSIVNN

KSDCKIQNSTGSFFWVNVKVNFDNVAMGYLALLQ

VATFKGWMDIMYAAVDSREVNMQPKWEDNVYM

YLYFVI

FIIFGGFFTLNLFVGVIIDNFNQQKKKLGGQDIFMTE

EQKKYYNAMKKLGSKKPQKPIPRPLNKFQGFVFDI

VTRQAFDITIMVLICLNMITMMVETDDQSEEKTKIL

GKINQFFVAVFTGECVMKMFALRQYYFTNGWNVF

DFIVVVLSIASLIFSAILKSLQSYFSPTLFRVIRLARIG

RILRLIRAAKGIRTLLFALMMSLPALFNIGLLLFLVM

FIYSIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCL

FQITTSAGWDGLLSPILNTGPPYCDPNLPNSNGTRG

DCGSPAVGIIFFTTYIIISFLIMVNMYIAVILENFNVA

TEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALS

DFADTLSGPLRIPKPNRNILIQMDLPLVPGDKIHCLD

ILFAFTKNVLGESGELDSLKANMEEKFMATNLSKS

SYEPIATTLRWKQEDISATVIQKAYRSYVLHRSMAL

SNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKS

ETASATSFPPSYESVTRGLSDRVNMRTSSSIQNEDE

ATSMELIAPGP

30
F0103262B06
EVQLVESGGGLVQPGGSLRLSCAGSTRTFSTYAMG

(parental)-FLAG-HIS6
WFRQAPGREREFVAHINFSGSSTRYADSVKGRFTIS

RDNAKNMGYLQMNSLKPEDTAVYYCAARWVAGP

PRYDYEYWGQGTLVTVSSAAADYKDHDGDYKDH

DIDYKDDDDKGAAHHHHHH

31
F0103262C02
EVQLVESGGGLVQAGGSLTLSCAASGLPFGLYILG

(parental)-FLAG-HIS6
WIRRAPGKERDFVAAISRSGRDTVYANSVKGRFTIS

RDNAKNMVYLRMDNLRPEDTAAYYCAVDSVPRG

TPTITESEYAIWGQGTLVTVSSAAADYKDHDGDYK

DHDIDYKDDDDKGAAHHHHHH

32
F0103265A11
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

(parental)-FLAG-HIS6
GWYRQAPGKQRELVAFIFSGGYTNYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSSAAADYKDHDGDYKDHDIDYKDDDDKG

AAHHHHHH

33
F0103265B04
EVQLVESGGGLVQPGGSLRLSCAAPSFIFSNNYME

(parental)-FLAG-HIS6
WYRQAPGKQRDWVARITGRGNTNYLDSVKGRFTI

SRDDAKNTVYLEIDSLKPEDTAVYYCSALWYGGR

AWGKGTLVTVSSAAADYKDHDGDYKDHDIDYKD

DDDKGAAHHHHHH

34
F0103275B05
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

(parental)-FLAG-HIS6
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRTYWGQGTLVTVSSAAADYKDHDGDYKDHDID

YKDDDDKGAAHHHHHH

35
F0103362B08
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

(parental)-FLAG-HIS6
WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSSAAADYKDHDGDY

KDHDIDYKDDDDKGAAHHHHHH

36
F0103387G04
EVQLVESGGGLAQPGGSLRLSCAASGPVFNINKMA

(parental)-FLAG-HIS6
WYRRAPGKQRELVASVTPTGSISYTDSVKGRFTISR

DGSKRWSLQMNSLTPEDTAVYYCNALLQPDSYSN

TRTYWGQGTLVTVSSAAADYKDHDGDYKDHDID

YKDDDDKGAAHHHHHH

37
F0103387G05
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

(parental)-FLAG-HIS6
WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSSAAADYKDHDGDYKD

HDIDYKDDDDKGAAHHHHHH

38
F0103454D07
EVQLVESGGGLVQPGGSLRLSCVASGGIININYIAW

(parental)-FLAG-HIS6
YRQTPGKQRDLVARISSDDTIKYGDSVKGRFAMSR

DKVKNMVHLQMNSLTTEDTGVYVCSALITPWTGD

TRTYWGRGTLVTVSSAAADYKDHDGDYKDHDID

YKDDDDKGAAHHHHHH

39
F0103464B09
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

(parental)-FLAG-HIS6
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSSAAADYKDHDGDYKD

HDIDYKDDDDKGAAHHHHHH

40
beta1 (beta1) subunit
MGRLLALVVGAALVSSACGGCVEVDSETEAVYGM

TFKILCISCKRRSETNAETFTEWTFRQKGTEEFVKIL

RYENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSI

FITNVTYNHSGDYECHVYRLLFFENYEHNTSVVKK

IHIEVVDKANRDMASIVSEIMMYVLIVVLTIWLVAE

MIYCYKKIAAATETAAQENASEYLAITSESKENCTG

VQVAE

41
beta2 (beta2) subunit
MHRDAWLPRPAFSLTGLSLFFSLVPPGRSMEVTVP

ATLNVLNGSDARLPCTFNSCYTVNHKQFSLNWTY

QECNNCSEEMFLQFRMKIINLKLERFQDRVEFSGNP

SKYDVSVMLRNVQPEDEGIYNCYIMNPPDRHRGH

GKIHLQVLMEEPPERDSTVAVIVGASVGGFLAVVIL

VLMVVKCVRRKKEQKLSTDDLKTEEEGKTDGEGN

PDDGAK

42
beta3 (beta3) subunit
MPAFNRLFPLASLVLIYWVSVCFPVCVEVPSETEAV

QGNPMKLRCISCMKREEVEATTVVEWFYRPEGGK

DFLIYEYRNGHQEVESPFQGRLQWNGSKDLQDVSI

TVLNVTLNDSGLYTCNVSREFEFEAHRPFVKTTRLI

PLRVTEEAGEDFTSVVSEIMMYILLVFLTLWLLIEMI

YCYRKVSKAEEAAQENASDYLAIPSENKENSAVPV

EE

43
P2A viral peptide
GSGATNFSLLKQAGDVEENPGP

44
huNav1.7-beta1
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

GTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRA

SILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPY

WIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH

HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDP

YEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRS

FRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLV

LAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRW

HMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQA

MCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLT

AIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAF

SKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGH

NFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLT

VTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRS

SSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG

CVRRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSW

FESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKI

FTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVD

VSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSR

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFYECINTTDGSRFPASQVPNRSECFALMN

VSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGW

TIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFG

SFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKK

YYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQ

AFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWI

NVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVI

ISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVK

GAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSA

GWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNP

SVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEEST

EPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAA

ALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILF

AFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYE

PITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISS

IYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSK

ESKKSGRGSGATNFSLLKQAGDVEENPGPMGRLLA

LVVGAALVSSACGGCVEVDSETEAVYGMTFKIL

CISCKRRSETNAETFTEWTFRQKGTEEFVKILRY

ENEVLQLEEDERFEGRVVWNGSRGTKDLQDLSI

FITNVTYNHSGDYECHVYRLLFFENYEHNTSVV

KKIHIEVVDKANRDMASIVSEIMMYVLIVVLTIW

LVAEMIYCYKKIAAATETAAQENASEYLAITSES

KENCTGVQVAE

45
muNav1.7
MAMLPPPGPQSFVHFTKQSLALIEQRISEEKAKGHK

DEKKDDEEEGPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKAIFRFNATPALY

MLSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTM

SNPPDWTKNVEYTFTGIYTFESLIKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRKDLEQNETLESIM

STAESEEELKRYFYYLEGSKDALLCGFSTDSGQCPE

GYECVTAGRNPDYGYTSFDTFGWAFLALFRLMTQ

DYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLIN

LILAVVAMAYEEQNQANIEEAKQKELEFQQMLDR

LKKEQEEAEAIAAAAAEYTSLGRSRIMGLSESSSET

SRLSSKSAKERRNRRKKKKQKLSSGEEKGDDEKLS

KSGSEESIRKKSFHLGVEGHHRAREKRLSTPNQSPL

SIRGSLFSARRSSRTSLFSFKGRGRDLGSETEFADDE

HSIFGDNESRRGSLFVPHRPRERRSSNISQASRSPPV

LPVNGKMHSAVDCNGVVSLVDGPSALMLPNGQLL

PEVIIDKATSDDSGTTNQMRKKRLSSSYFLSEDMLN

DPHLRQRAMSRASILTNTVEELEESRQKCPPWWYR

FAHTFLIWNCSPYWIKFKKFIYFIVMDPFVDLAITICI

VLNTLFMAMEHHPMTDEFKNVLAVGNLVFTGIFA

AEMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELF

LADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIG

NSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKECV

CKINENCKLPRWHMNDFFHSFLIVFRVLCGEWIET

MWDCMEVAGQTMCLIVYMMVMVIGNLVVLNLFL

ALLLSSFSSDNLTAIEEDTDANNLQIAVARIKRGINY

VKQTLREFILKSFSKKPKGSKDTKRTADPNNKREN

YISNRTLAEISKDHNFLKEKDKISGFSSSLDKSFMDE

NDYQSFIHNPSLTVTVPIAPGESDLENMNTEELSSDS

DSDYSKERRNRSSSSECSTVDNPLPGEEEAEAEPIN

ADEPEACFTDGCVRRFPCCQVNIDTGKGKVWWTIR

KTCYRIVEHSWFESFIVLMILLSSGALAFEDIYIEKK

KTIKIILEYADKIFTYIFILEMLLKWVAYGYKTYFTN

AWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRT

LRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLLV

CLIFWLIFSIMGVNLFAGKFYECVNTTDGSRFSVSQ

VANRSECFALMNVSGNVRWKNLKVNFDNVGLGY

LSLLQVATFKGWMDIMYAAVDSVNVNAQPIYEYN

LYMYIYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKLG

GQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNK

FQGCIFDLVTNQAFDITIMVLICLNMVTMMVEKEG

QTDYMSFVLYWINVVFIILFTGECVLKLISLRHYYF

TVGWNIFDFVVVILSIVGMFLAEMIEKYFVSPTLFR

VIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNI

GLLLFLVMFIYAIFGMSNFAYVKKEAGINDMFNFE

TFGNSMICLFQITTSAGWDGLLAPILNSAPPDCDPK

KVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNM

YIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFD

PDATQFIEFCKLSDFAAALDPPLLIAKPNKVQLIAM

DLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQ

MEERFMSANPSKVSYEPITTTLKRKQEDVSATIIQR

AYRRYRLRQNVKNISSIYIKDGDRDDDLPNKEDIVF

DNVNENSSPEKTDATASTISPPSYDSVTKPDQEKYE

TDKTEKEDKEKDESRK

46
F0103262B06 (parental)
EVQLVESGGGLVQPGGSLRLSCAGSTRTFSTYAMG

WFRQAPGREREFVAHINFSGSSTRYADSVKGRFTIS

RDNAKNMGYLQMNSLKPEDTAVYYCAARWVAGP

PRYDYEYWGQGTLVTVSS

47
F0103262C02 (parental)
EVQLVESGGGLVQAGGSLTLSCAASGLPFGLYILG

WIRRAPGKERDFVAAISRSGRDTVYANSVKGRFTIS

RDNAKNMVYLRMDNLRPEDTAAYYCAVDSVPRG

TPTITESEYAIWGQGTLVTVSS

48
F0103265A11 (parental)
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGYTNYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

49
F0103265B04 (parental)
EVQLVESGGGLVQPGGSLRLSCAAPSFIFSNNYME

WYRQAPGKQRDWVARITGRGNTNYLDSVKGRFTI

SRDDAKNTVYLEIDSLKPEDTAVYYCSALWYGGR

AWGKGTLVTVSS

50
F0103275B05 (parental)
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

51
F0103362B08 (parental)
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

52
F0103387G04 (parental)
EVQLVESGGGLAQPGGSLRLSCAASGPVFNINKMA

WYRRAPGKQRELVASVTPTGSISYTDSVKGRFTISR

DGSKRWSLQMNSLTPEDTAVYYCNALLQPDSYSN

TRTYWGQGTLVTVSS

53
F0103387G05 (parental)
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

54
F0103454D07 (parental)
EVQLVESGGGLVQPGGSLRLSCVASGGIININYIAW

YRQTPGKQRDLVARISSDDTIKYGDSVKGRFAMSR

DKVKNMVHLQMNSLTTEDTGVYVCSALITPWTGD

TRTYWGRGTLVTVSS

55
F0103464B09 (parental)
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

56
FLAG-HIS6 peptide
AAADYKDHDGDYKDHDIDYKDDDDKGAAHHHH

HH

57
human VH3-JH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMH

consensus (amino acids
WVRQAPGKGLEWVSVISSSGSSTYYADSVKGRFTI

Xaa99-Xaal 14 are each
SRDNSKNTLYLQMNSLRAEDTAVYYCARXXXXXX

independently any
XXXXXXXXXXWGQGTLVTVSS

amino acid except Cys)

58
VHH2-consensus
QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMG

WYRQAPGKQRELVAAITSGGSTNYADSVKGRFTIS

RDNAKNTLYLQMNSLKPEDTAVYYCNA

59
F0103387G04_SO
DVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMAWY

RQAPGKQRELVAYVTPTGDISYADSVKGRFTISDDGSKR

VSLQMNSLRPEDTALYYCRALLQPSSYSGTRTYWGQGT

LVTVSS

60
F0103387G05_SO
DVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMRWYR

QAPGKQREFVARITGGSATGYADSVKGRFTISRDNAKN

TVYLQMNSLRPEDTALYYCEALVTASVRGGSIHSGTYW

GQGTLVTVSS

61
F0103464B09_SO
DVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFTGWY

RQAPGKQRELVARIYEGGNTQYADFAKGRFSISRDNAK

KTVYLQMNSLRAEDTALYYCLFSGTISTGREYRSGDYW

GQGTLVTVSS

62
Nav1.7 alpha epitope
FRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKD

ALLCGFSTDSGQCPEGYTCV

63
Nav1.7 alpha Domain I
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPK

EEKKDDDEEAPKPSSDLEAGKQLPFIYGDIPPGMVS

EPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYM

LSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMN

NPPDWTKNVEYTFTGIYTFESLVKILARGFCVGEFT

FLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFR

VLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

CLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIM

NTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPE

GYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQD

YWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLI

LAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLK

KEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKS

ESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIR

GSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSI

FGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLP

VNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPE

VIIDKATSDDSGTTNQIHKKRRCSSYLLSEDMLNDP

NLRQRAMSRASILTNTVEELEESRQKCPPWWYRFA

HKFLIWNCSPYWIKFKKCIY

64
Nav1.7alpha Domain I
KHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLE

S5-S6 loop
GSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGYT

SFDTFSWAFLALFRLMTQDYWENLYQQTLRAAGK

TY

65
Nav1.7 alpha Exon 5N
YLTEFVNLGNVS

66
Nav1.7 alpha Exon 5A
YVTEFVDLGNVS

67
Nav1.7 alpha Exon 11S
LPNGQLLPE

68
Nav1.7 alpha Exon 11L
LPNGQLLPEVIIDKATSDDS

69
>F010301461
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINRMA

F0103275B05(S33R, S50Y,
WYRRAPGKQRELVAYSTNGGDTNYADSVKGRFTI

S56D, N93R)
SRDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYD

ISRTYWGQGTLVTVSS

70
>F010301635
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(L11V, R39Q,
WYRQAPGKQRELVASSTNGGSTNYADSVKGRFTIS

T83R, V89L)
RDNAKRVYLQMNSLRPEDTALYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

71
>F010301636
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(L11V ,R76N,
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

T83R, V89L)
RDNAKNVYLQMNSLRPEDTALYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

72
>F010301637
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(L11V, T83R,
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

V89L)
RDNAKRVYLQMNSLRPEDTALYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

73
>F010301638
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(L11V, R39Q,
WYRQAPGKQRELVASSTNGGSTNYADSVKGRFTIS

R76N, T83R, V89L)
RDNAKNVYLQMNSLRPEDTALYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

74
>F010301639
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(R76_V78
WYRQAPGKQRELVASSTNGGSTNYADSVKGRFTIS

insT)(L11V, R39Q, T83R,
RDNAKRTVYLQMNSLRPEDTALYYCNALLQPSIYD

V89L)
ISRTYWGQGTLVTVSS

75
>F010301640
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(R76_V78
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

insT)(L11V, T83R, V89L)
RDNAKRTVYLQMNSLRPEDTALYYCNALLQPSIYD

ISRTYWGQGTLVTVSS

76
>F010301641
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(R76_V78
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

insT)(L11V, R76N, T83R,
RDNAKNTVYLQMNSLRPEDTALYYCNALLQPSIYD

V89L)
ISRTYWGQGTLVTVSS

77
>F010301642
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINSMA

F0103275B05(R76_V78
WYRQAPGKQRELVASSTNGGSTNYADSVKGRFTIS

insT)(L11V, R39Q, R76N,
RDNAKNTVYLQMNSLRPEDTALYYCNALLQPSIYD

T83R, V89L)
ISRTYWGQGTLVTVSS

78
>F010301652
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINRMA

F0103275B05(L11V, S33R,
WYRRAPGKQRELVAYSTNGGDTNYADSVKGRFTI

S50Y, S56D, R76N, T83R,
SRDNAKNVYLQMNSLRPEDTALYYCRALLQPSIYD

V89L, N93R)
ISRTYWGQGTLVTVSS

79
>F010301653
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINRMA

F0103275B05(L11V, S33R,
WYRRAPGKQRELVAYSTNGGDTNYADSVKGRFTI

S50Y, S56D, T83R, V89L,
SRDNAKRVYLQMNSLRPEDTALYYCRALLQPSIYD

N93R)
ISRTYWGQGTLVTVSS

80
>F010301654
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINRMA

F0103275B05(L11V, S33R,
WYRQAPGKQRELVAYSTNGGDTNYADSVKGRFTI

R39Q, S50Y, S56D, T83R,
SRDNAKRVYLQMNSLRPEDTALYYCRALLQPSIYD

V89L, N93R)
ISRTYWGQGTLVTVSS

81
>F010301655
EVQLVESGGGVVQPGGSLRLSCAASGSIFNINRMA

F0103275B05(L11V, S33R,
WYRQAPGKQRELVAYSTNGGDTNYADSVKGRFTI

R39Q, S50Y, S56D, R76N,
SRDNAKNVYLQMNSLRPEDTALYYCRALLQPSIYD

T83R, V89L, N93R)
ISRTYWGQGTLVTVSS

82
>F010301556
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

F0103387G05(D23A, D53G,
WHRQGAGKQREFVARITGGSATGYADSVKGRFTIS

D54G, D58G)
RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

83
>F010301563
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

F0103387G05(D23A, D58G)
WHRQGAGKQREFVARITDDSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

84
>F010301849
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQAPGKQREFVARITDDSATGYADSVKGRFTIS

D23A, G40A, A41P, D58G,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

N82bS, N83R, V89L,
GGSIHSGTYWGQGTLVTVSS

R105Q)

85
>F010301850
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITDDSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P, D58G, N82bS, N83R,
GGSIHSGTYWGQGTLVTVSS

V89L, R105Q)

86
>F010301643
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

D23A, N82bS, N83R,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

V89L, R105Q)
GGSIHSGTYWGQGTLVTVSS

87
>F010301644
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQGAGKQREFVARITDDSATDYADSVKGRFTIS

H37Y, N82bS, N83R,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

V89L, R105Q)
GGSIHSGTYWGQGTLVTVSS

88
>F010301645
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQAAGKQREFVARITDDSATDYADSVKGRFTIS

G40A, N82bS, N83R,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

V89L, R105Q)
GGSIHSGTYWGQGTLVTVSS

89
>F010301646
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQGPGKQREFVARITDDSATDYADSVKGRFTIS

A41P, N82bS, N83R,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

V89L, R105Q)
GGSIHSGTYWGQGTLVTVSS

90
>F010301647
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQGAGKQRELVARITDDSATDYADSVKGRFTIS

F47L, N82bS, N83R,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

V89L, R105Q)
GGSIHSGTYWGQGTLVTVSS

91
>F010301648
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

N82bS, N83R, V89L,
RDNAKNTVYLQMNSLRPEDTALYYCNALVTASVR

E93N, R105Q)
GGSIHSGTYWGQGTLVTVSS

92
>F010301649
EVQLVESGGGVVQPGGSLRLSCDASGRILRIGYMR

F0103387G05(L11V, A14P,
WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

N82bS, N83R, V89L,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

R105Q)
GGSIHSGTYWGQGTLVTVSS

93
>F010302307
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITDDSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDAAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P, D58G, N73A, N82bS,
GGSIHSGTYWGQGTLVTVSS

N83R, V89L, R105Q)

94
>F010302308
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITDDSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDYAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P, D58G, N73Y, N82bS,
GGSIHSGTYWGQGTLVTVSS

N83R, V89L, R105Q)

95
>F010302309
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITDDSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDQAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P, D58G, N73Q, N82bS,
GGSIHSGTYWGQGTLVTVSS

N83R, V89L, R105Q)

96
>F010302391
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITGGSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDNAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P,D53G, D54G, D58G,
GGSIHSGTYWGQGTLVTVSS

N82bS, N83R, V89L,

R105Q)

97
>F010302392
EVQLVESGGGVVQPGGSLRLSCAASGRILRIGYMR

F0103387G05(L11V, A14P,
WYRQAPGKQREFVARITGGSATGYADSVKGRFTIS

D23A, H37Y, G40A,
RDQAKNTVYLQMNSLRPEDTALYYCEALVTASVR

A41P, D53G, D54G, D58G,
GGSIHSGTYWGQGTLVTVSS

N73Q, N82bS, N83R,

V89L, R105Q)

98
>F010301868
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S68T,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFTIS

T79Y, R81Q, S82aN,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

N82bS, K83R, G88A,
REYRSGDYWGQGTLVTVSS

V89L)

99
>F010301869
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S68T,
WYRRVPGKERELVARIYNGGNTNY

M77T, T79Y, R81Q,
ADFAKGRFTISRDNAKKTVYLQMNSLRPEDTALYY

S82aN, N82bS, K83R, G88A,
CLFSGTINTGREYRSGDYWGQGTLVTVSS

V89L)

100
>F010301870
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S68T,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFTIS

T79Y, R81Q, S82aN,
RDNAKKMVYLQMNSLRPEDTALYYCNFSGTINTG

N82bS, K83R, G88A, V89L,
REYRSGDYWGQGTLVTVSS

L93N)

101
>F010301871
EVQLVESGGGVVQPGGSLRLSCAATSRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRVPGKERELVARIYNGGNTNYADFAKGRFT

S68T, T79Y, R81Q,
ISRDNAKKMVYLQMNSLRPEDTALYYCLFSGTINT

S82aN, N82bS, K83R,
GREYRSGDYWGQGTLVTVSS

AG88, V89L)

102
>F010301872
EVQLVESGGGVVQPGGSLRLSCATSSRAFIRDVFTG

F0103464B09(L11V, T25S,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

103
>F010301873
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, R39Q,
WYRQVPGKERELVARIYNGGNTNYADFAKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

104
>F010301874
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, V40A,
WYRRAPGKERELVARIYNGGNTNYADFAKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

105
>F010301875
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, F62S,
WYRRVPGKERELVARIYNGGNTNYADSAKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

106
>F010301876
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, A63V,
WYRRVPGKERELVARIYNGGNTNYADFVKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

107
>F010301877
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S68T,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFTIS

K76N, T79Y, R81Q,
RDNAKNMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

108
>F010301892
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDLFTG

F0103464B09(V33L)
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

109
>F010301893
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, E44Q,
WYRRVPGKQRELVARIYNGGNTNYADFAKGRFTIS

S68T, T79Y, R81Q,
RDNAKKMVYLQMNSLRPEDTALYYCLFSGTINTG

S82aN, N82bS, K83R,
REYRSGDYWGQGTLVTVSS

G88A, V89L)

110
>F010301932
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, K83R,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

V89L)
RDNAKKMVTLRMSNLRPEDTGLYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

111
>F010301933
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S68T,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFTIS

K83R, V89L)
RDNAKKMVTLRMSNLRPEDTGLYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

112
>F010301934
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, M77T,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

K83R, V89L)
RDNAKKTVTLRMSNLRPEDTGLYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

113
>F010301935
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, T79Y,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

K83R, V89L)
RDNAKKMVYLRMSNLRPEDTGLYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

114
>F010301936
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, R81Q,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

K83R, V89L)
RDNAKKMVTLQMSNLRPEDTGLYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

115
>F010301937
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, S82aN,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

K83R, V89L)
RDNAKKMVTLRMNNLRPEDTGLYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

116
>F010301938
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, N82bS,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

K83R, V89L)
RDNAKKMVTLRMSSLRPEDTGLYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

117
>F010301939
EVQLVESGGGVVQPGGSLRLSCATTSRAFIRDVFTG

F0103464B09(L11V, K83R,
WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

G88A, V89L)
RDNAKKMVTLRMSNLRPEDTALYYCLFSGTINTGR

EYRSGDYWGQGTLVTVSS

118
>F010302333
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTNYADSAKGRFT

T25S, A28Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, G54E, F62S, S68T,
REYRSGDYWGQGTLVTVSS

M77T, T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

119
>F010302334
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTQYADSAKGRFT

T25S, A28Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, G54E, N58Q, F62S,
REYRSGDYWGQGTLVTVSS

S68T, M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

120
>F010302335
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTQYADSAKGRFTI

T25S, A28Q, V40A,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, G54S, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

121
>F010302336
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTQYADSAKGRFT

T25S, S26H, A28Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, G54E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

122
>F010302337
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYEGGNTQYADSAKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

123
>F010302338
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTQYADSAKGRFTI

T25S, S26H, V40A,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, G54S, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

124
>F010302339
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYEGGNTQYADSAKGRFT

T25S, S26H, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, N58Q, F62S,
REYRSGDYWGQGTLVTVSS

S68T, M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

125
>F010302340
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDLFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTNYADSAKGRFTI

T25S, S26H, V33L,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

126
>F010302341
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTNYADSAKGRFT

T25S, A28Q, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, G54E, F62S,
REYRSGDYWGQGTLVTVSS

S68T, M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

127
>F010302342
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTQYADSAKGRFT

T25S, A28Q, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, G54E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

128
>F010302343
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTQYADSAKGRFTI

T25S, A28Q, R39Q, V40A,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

129
>F010302344
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTQYADSAKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, G54E,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

130
>F010302345
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYEGGNTQYADSAKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, N53E,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

131
>F010302346
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTQYADSAKGRFTI

T25S, S26H, R39Q,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

132
>F010302347
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYEGGNTQYADSAKGRFT

T25S, S26H, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

133
>F010302348
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDLFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTNYADSAKGRFTI

T25S, S26H, V33L,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, N53E,
REYRSGDYWGQGTLVTVSS

G54S,F62S, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

134
>F010302349
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTNYADSVKGRFT

T25S, A28Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, G54E, F62S, A63V,
REYRSGDYWGQGTLVTVSS

S68T, M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

135
>F010302350
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTQYADSVKGRFT

T25S, A28Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, G54E, N58Q, F62S,
REYRSGDYWGQGTLVTVSS

A63V, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

136
>F010302351
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTQYADSVKGRFTI

T25S, A28Q, V40A,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, G54S, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN, N82bS,

K83R, G88A,

V89L, N99S)

137
>F010302352
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNEGNTQYADSVKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, G54E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

138
>F010302353
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYEGGNTQYADSVKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

139
>F010302354
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTQYADSVKGRFTI

T25S, S26H, V40A,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, G54S, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

140
>F010302355
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYEGGNTQYADSVKGRFT

T25S, S26H, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, N53E, N58Q, F62S,
REYRSGDYWGQGTLVTVSS

A63V, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

141
>F010302356
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDLFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYESGNTNYADSVKGRFTI

T25S, S26H, V33L,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

142
>F010302357
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTNYADSVKGRFT

T25S, A28Q, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, G54E, F62S,
REYRSGDYWGQGTLVTVSS

A63V, S68T, M77T, T79Y,

R81Q, S82aN, N82bS,

K83R, G88A, V89L,

N99S)

143
>F010302358
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTQYADSVKGRFT

T25S, A28Q, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, G54E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

144
>F010302359
EVQLVESGGGVVQPGGSLRLSCAASSRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTQYADSVKGRFTI

T25S, A28Q, R39Q,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, A63V, S68T,

M77T, T79Y, R81Q, S82aN,

N82bS, K83R,

G88A, V89L, N99S)

145
>F010302360
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNEGNTQYADSVKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, G54E,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, A63V, S68T,

M77T, T79Y, R81Q, S82aN,

N82bS, K83R,

G88A, V89L, N99S)

146
>F010302361
EVQLVESGGGVVQPGGSLRLSCAASHRQFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYEGGNTQYADSVKGRFT

T25S, S26H, A28Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, N53E,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, A63V, S68T,

M77T, T79Y, R81Q, S82aN,

N82bS, K83R,

G88A, V89L, N99S)

147
>F010302362
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTQYADSVKGRFTI

T25S, S26H, R39Q,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, G54S,
REYRSGDYWGQGTLVTVSS

N58Q, F62S, A63V, S68T,

M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

148
>F010302363
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYEGGNTQYADSVKGRFT

T25S, S26H, R39Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

V40A, E44Q, N53E, N58Q,
REYRSGDYWGQGTLVTVSS

F62S, A63V, S68T, M77T,

T79Y, R81Q, S82aN,

N82bS, K83R, G88A,

V89L, N99S)

149
>F010302364
EVQLVESGGGVVQPGGSLRLSCAASHRAFIRDLFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYESGNTNYADSVKGRFTI

T25S, S26H, V33L,
SRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

R39Q, V40A, E44Q, N53E,
REYRSGDYWGQGTLVTVSS

G54S, F62S, A63V, S68T,

M77T, T79Y, R81Q,

S82aN, N82bS, K83R,

G88A, V89L, N99S)

150
>F010302365
EVQLVESGGGVVQPGGSLRLSCAASSRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNGGNTNYADSAKGRFT

T25S, V40A, E44Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

F62S, S68T, M77T, T79Y,
REYRSGDYWGQGTLVTVSS

R81Q, S82aN, N82bS,

K83R, G88A, V89L, N99S)

151
>F010302366
EVQLVESGGGVVQPGGSLRLSCAASSRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNGGNTNYADSAKGRFT

T25S, R39Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, F62S, S68T, M77T,
REYRSGDYWGQGTLVTVSS

T79Y, R81Q, S82aN,

N82bS, K83R, G88A, V89L,

N99S)

152
>F010302367
EVQLVESGGGVVQPGGSLRLSCAASSRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRRAPGKQRELVARIYNGGNTNYADSVKGRFT

T25S, V40A, E44Q,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

F62S, A63V, S68T, M77T,
REYRSGDYWGQGTLVTVSS

T79Y, R81Q, S82aN,

N82bS, K83R, G88A, V89L,

N99S)

153
>F010302368
EVQLVESGGGVVQPGGSLRLSCAASSRAFIRDVFT

F0103464B09(L11V, T24A,
GWYRQAPGKQRELVARIYNGGNTNYADSVKGRFT

T25S, R39Q, V40A,
ISRDNAKKTVYLQMNSLRPEDTALYYCLFSGTISTG

E44Q, F62S, A63V, S68T,
REYRSGDYWGQGTLVTVSS

M77T, T79Y, R81Q,

S82aN, N82bS, K83R, G88A,

V89L,N99S)

154
>F010301656
EVQLVESGGGLAQPGGSLRLSCAASGPVFNINRMA

F0103387G04(K33R, S50Y,
WYRRAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

S56D, N93R)
RDGSKRWSLQMNSLTPEDTAVYYCRALLQPDSYS

NTRTYWGQGTLVTVSS

155
>F010301840
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(R76_V7
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

8insT)(L11V, A12V, K33R,
RDGSKRTWSLQMNSLRPEDTALYYCRALLQPDSYS

R39Q, S50Y, S56D,
NTRTYWGQGTLVTVSS

T83R, V89L, N93R)

156
>F010301841
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRWSLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T83R, V89L, N93R)
NTRTYWGQGTLVTVSS

157
>F010301842
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRWSLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, T83R, V89L,
NTRTYWGQGTLVTVSS

N93R)

158
>F010301843
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

K33R, R39Q, S50Y,
RDNSKRWSLQMNSLRPEDTALYYCRALLQPDSYS

S56D, G73N, T83R, V89L,
NTRTYWGQGTLVTVSS

N93R)

159
>F010301844
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKNWSLQMNSLRPEDTALYYCRALLQPDSYS

S56D, R76N, T83R, V89L,
NTRTYWGQGTLVTVSS

N93R)

160
>F010301845
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVSLQMNSLRPEDTALYYCRALLQPDSYSN

S56D, W78V, T83R, V89L,
TRTYWGQGTLVTVSS

N93R)

161
>F010301846
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYTDSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRWYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, S79Y, T83R, V89L,
NTRTYWGQGTLVTVSS

N93R)

162
>F010301847
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDNSKNVYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, G73N, R76N,
NTRTYWGQGTLVTVSS

W78V, S79Y, T83R, V89L,

N93R)

163
>F010301848
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDNSKRVYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, G73N, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R)

164
>F010301865
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, W78V, S79Y,
NTRTYWGQGTLVTVS

T83R, V89L, N93R)

165
>F010301866
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKNVYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, R76N, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R)

166
>F010302310
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPRRYS

S56D, T60A, G73A, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, S100R)-

FLAG3-HIS6

167
>F010302311
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPRRYS

S56D, T60A, G73R, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, S100R)-

FLAG3-HIS6

168
>F010302312
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPDSYSI

S56D, T60A, W78V, S79Y,
TRTYWGQGTLVTVSS

T83R, V89L, N93R, N100cI)

169
>F010302313
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPDSYSI

S56D, T60A, G73A, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

N100cI)

170
>F010302314
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPDSYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

N100cI)

171
>F010302315
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPRSYSI

S56D, T60A, W78V, S79Y,
TRTYWGQGTLVTVSS

T83R, V89L, N93R, D99R,

N100cI)

172
>F010302316
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPRSYSI

S56D, T60A, G73A, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, N100cI)-

FLAG3-HIS6

173
>F010302317
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPRSYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, N100cI)-

FLAG3-HIS6

174
>F010302318
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPDRYSI

S56D, T60A, W78V, S79Y,
TRTYWGQGTLVTVSS

T83R, V89L, N93R, S100R,

N100cI)

175
>F010302319
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPDRYSI

S56D, T60A, G73A, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

S100R, N100cI)-

FLAG3-HIS6

176
>F010302320
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPDSYS

S56D, T60A, G73A, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R)

177
>F010302321
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPDRYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

S100R,N100cI)-

FLAG3-HIS6

178
>F010302322
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, 50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPRRYSI

S56D, T60A, W78V, S79Y,
TRTYWGQGTLVTVSS

T83R, V89L, N93R, D99R,

S100R, N100cI)-

FLAG3-HIS6

179
>F010302323
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPRRYSI

S56D, T60A, G73A, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, S100R, N100cI)

180
>F010302324
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPRRYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99R, S100R, N100cI)

181
>F010302325
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPDSYSN

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R)

182
>F010302326
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V,A1
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

2V,K33R,R39Q,S50Y,S
RDGSKRVYLQMNSLRPEDTALYYCRALLQPRSYSN

56D,T60A, W78V,S79Y
TRTYWGQGTLVTVSS

T83R, V89L,N93R,D99

R)

183
>F010302327
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V,A1
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

2V,K33R,R39Q,S50Y,S
RDASKRVYLQMNSLRPEDTALYYCRALLQPRSYSN

56D,T60A, G73A, W78V
TRTYWGQGTLVTVSS

,S79Y,T83R,V89L,N93

R,D99R)

184
>F010302328
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V,A1
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

2V,K33R,R39Q,S50Y,S
RDRSKRVYLQMNSLRPEDTALYYCRALLQPRSYSN

56D,T60A,G73R, W78V
TRTYWGQGTLVTVSS

,S79Y,T83R,V89L,N93

R,D99R)

185
>F010302329
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V,A1
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

2V,K33R,R39Q,S50Y,S
RDGSKRVYLQMNSLRPEDTALYYCRALLQPDRYS

56D,T60A,W78V,S79Y
NTRTYWGQGTLVTVSS

T83R,V89L,N93R,S10

0R)

186
>F010302330
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDASKRVYLQMNSLRPEDTALYYCRALLQPDRYS

5S6D, T60A, G73A, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

S100R)

187
>F010302331
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPDRYS

S56D, T60A, G73R, W78V,
NTRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

S100R)

188
>F010302332
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDGSKRVYLQMNSLRPEDTALYYCRALLQPRRYS

S56D, T60A, W78V, S79Y,
NTRTYWGQGTLVTVSS

T83R, V89L, N93R, D99R,

S100R)

189
>F010302370
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPSSYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99S, N100cI)

190
>F010302371
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPNVYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99N, S100V, N100cI)

191
>F010302372
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RDRSKRVYLQMNSLRPEDTALYYCRALLQPDVYSI

S56D, T60A, G73R, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

S100V, N100cI)

192
>F010302383
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RGGSKRVYLQMNSLRPEDTALYYCRALLQPSSYSG

S56D, T60A, D72G, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99S, N100cG)

193
>F010302384
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RGGSKRVYLQMNSLRPEDTALYYCRALLQPSSYSI

S56D, T60A, D72G, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99S, N100cI)

194
>F010302385
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RQGSKRVYLQMNSLRPEDTALYYCRALLQPSSYSG

S56D, T60A, D72Q, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99S, N100cG)

195
>F010302386
EVQLVESGGGVVQPGGSLRLSCAASGPVFNINRMA

F0103387G04(L11V, A12V,
WYRQAPGKQRELVAYVTPTGDISYADSVKGRFTIS

K33R, R39Q, S50Y,
RQGSKRVYLQMNSLRPEDTALYYCRALLQPSSYSI

S56D, T60A, D72Q, W78V,
TRTYWGQGTLVTVSS

S79Y, T83R, V89L, N93R,

D99S, N100cI)

196
F0103275B05-CDR1
GSIFNINSMA

197
F0103275B05-CDR1-A
GSIFNINRMA

198
F0103275B05-CDR2
SSTNGGSTN

199
F0103275B05-CDR2-A
YSTNGGDTN

200
F0103275B05-CDR3
LLQPSIYDISRTY

201
F0103387G05-CDR1
GRILRIGYMR

202
F0103387G05-CDR2
RITDDSATD

203
F0103387G05-CDR2-A
RITGGSATG

204
F0103387G05-CDR2-B
RITDDSATG

205
F0103387G05-CDR2-C
RITGGSATG

206
F0103387G05-CDR3
LVTASVRGGSIHSGTY

207
F0103464B09-CDR1
SRAFIRDVFTG

208
F0103464B09-CDR1-A
SRAFIRDLFTG

209
F0103464B09-CDR1-B
SRQFIRDVFTG

210
F0103464B09-CDR1-C
HRQFIRDVFTG

211
F0103464B09-CDR1-D
HRAFIRDVFTG

212
F0103464B09-CDR1-E
HRAFIRDLFTG

213
F0103464B09-CDR2
RIYNGGNTN

214
F0103464B09-CDR2-A
RIYNEGNTN

215
F0103464B09-CDR2-B
RIYNEGNTQ

216
F0103464B09-CDR2-C
RIYESGNTQ

217
F0103464B09-CDR2-D
RIYESGNTN

218
F0103464B09-CDR2-E
RIYNEGNTN

219
F0103464B09-CDR3
SGTINTGREYRSGDY

220
F0103464B09-CDR3-A
SGTISTGREYRSGDY

221
F0103387G04-CDR1
GPVFNINKMA

222
F0103387G04-CDR1-A
GPVFNINRMA

223
F0103387G04-CDR2
SVTPTGSIS

224
F0103387G04-CDR2-A
YVTPTGDIS

225
F0103387G04-CDR3
LLQPDSYSNTRTY

226
F0103387G04-CDR3-A
LLQPRRYSNTRTY

227
F0103387G04-CDR3-B
LLQPDSYSITRTY

228
F0103387G04-CDR3-C
LLQPRSYSITRTY

229
F0103387G04-CDR3-B
LLQPRSYSNTRTY

230
F0103387G04-CDR3-E
LLQPSSYSITRTY

231
F0103387G04-CDR3-F
LLQPNVYSITRTY

232
F0103387G04-CDR3-G
LLQPDVYSITRTY

233
F0103387G04-CDR3-H
LLQPSSYSGTRTY

234
ALB11002 (“ALB201”)
EVQLVESGGGXVQPGNSLRLSCAASGFTFSSFGMS

without C-
WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

terminal A, X11 is L or
SRDNAKTTLYLQMNSLRPEDTAXYYCTIGGSLSRS

V, X93 is V or L
SQGTLVTVSS

235
HSA-CDR1
GFTFSSFGMS

236
HSA-CDR2
SISGSGSDTL

237
HSA-CDR3
GGSLSR

238
ALB11002 (“ALB201”),
EVQLVESGGGXVQPGNSLRLSCAASGFTFSSFGMS

X11 is L or
WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

V, X93 is V or L
SRDNAKTTLYLQMNSLRPEDTAXYYCTIGGSLSRS

SQGTLVTVSSA

239
ALB11002 (“ALB201”)
DVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMS

E1D L11V
WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

L93V without C-
SRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSS

terminal A
QGTLVTVSS

240
ALB11002 (“ALB201”)
DVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMS

E1D L11V
WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

L93V
SRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSS

QGTLVTVSSA

241
AB11 without C-
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMS

terminal A
WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS

SQGTLVTVSS

242
AB11
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMS

WVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI

SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRS

SQGTLVTVSS

243
9GS-linker
GGGGSGGGS

244
20GS linker
GGGGSGGGGSGGGGSGGGGS

245
35GS linker
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG

GS

246
Linker subunit
GGGS

247
F0103262B06-CDR1
TRTFSTYAMG

248
F0103262B06-CDR2
HINFSGSSTRY

249
F0103262B06-CDR3
ARWVAGPPRYDYEY

250
F0103262C02-CDR1
GLPFGLYILG

251
F0103262C02-CDR2
AISRSGRDTV

252
F0103262C02-CDR3
DSVPRGTPTITESEYAI

253
F0103265A11-CDR1
GMLFNANTQG

254
F0103265A11-CDR2
FIFSGGYTN

255
F0103265A11-CDR3
SRY

256
F0103265B04-CDR1
SFIFSNNYME

257
F0103265B04-CDR2
RITGRGNTN

258
F0103265B04-CDR3
LWYGGRA

259
F0103362B08-CDR1
VRPFSTSAMG

260
F0103362B08-CDR2
GILWNGIVTY

261
F0103362B08-CDR3
DRDYGGRSFSAYEYEY

262
F0103454D07-CDR1
GGIININYIA

263
F0103454D07-CDR2
RISSDDTIK

264
F0103454D07-CDR3
LITPWTGDTRTY

265
ALB00223
EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMS

WVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTI

SRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSS

QGTLVTVSSA

266
ALB00223 without C-
EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMS

terminal A
WVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTI

SRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSS

QGTLVTVSS

267
ALB00223-HSA-CDR1
GFTFRSFGMS

268
VHH2-consensus
QVQLVESGGGLVQAGGSLRLSCAAS

Framework 1

269
VHH2-consensus
WYRQAPGKQRELVA

Framework 2

270
VHH2-consensus
YADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVY

Framework 3
YCNA

271
VHH2-consensus
WGQGTLVTVSS

Framework 4

272
Nucleotide sequence
GCGGCCGCAGATTATAAAGATCATGATGGCGATT

encoding FLAG-HIS6
ATAAAGATCATGATATTGATTATAAAGATGATGA

peptide
TGATAAAGGGGCCGCACATCATCATCATCATCAT

273
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGTTTG

encoding F0103262B06
GTGCAGCCTGGGGGCTCTCTGAGACTCTCGTGTG

CTGGGTCTACACGCACGTTTAGCACCTATGCCAT

GGGCTGGTTCCGCCAGGCTCCAGGGAGGGAGCG

TGAGTTTGTAGCACATATTAATTTTAGCGGTAGT

AGCACAAGGTATGCAGACTCCGTGAAGGGCCGA

TTCACCATCTCCAGAGACAACGCCAAGAATATGG

GATATCTGCAGATGAATAGCCTGAAACCTGAGG

ACACGGCCGTTTATTATTGTGCAGCCCGGTGGGT

CGCTGGCCCTCCGAGGTATGACTATGAGTACTGG

GGCCAGGGGACCCTGGTCACCGTCTCCTCA

274
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTG

encoding F0103262C02
GTGCAGGCTGGGGGCTCTCTGACACTCTCCTGTG

CAGCCTCTGGTCTGCCCTTCGGATTGTATATTCTG

GGCTGGATCCGCCGGGCTCCAGGGAAGGAGCGT

GATTTTGTAGCAGCTATTAGCCGGAGTGGTAGGG

ACACGGTTTATGCAAACTCCGTGAAGGGCCGATT

CACCATCTCCAGAGACAACGCCAAGAACATGGT

GTACCTGCGAATGGACAATCTGAGACCGGAGGA

CACGGCCGCATATTACTGTGCAGTGGACTCAGTG

CCACGCGGAACTCCTACCATCACAGAGTCTGAGT

ACGCCATCTGGGGCCAGGGGACCCTGGTCACCGT

CTCCTCA

275
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103265A11
GTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAATGCTCTTCAACGCCAATACCCA

GGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATTTATTTTTAGTGGTGGTTAC

ACAAACTATGTAGACTCCGTGAAGGGCCGTTTCA

CCATCTCCAGAGACAACGCCAAGCGCACAATGT

ATCTGCAGATGAACAGCCTGAAACCTGAGGACT

CGGCCATCTATTACTGCTCATTGAGTCGCTACTT

GGGCCAGGGGACCCTGGTCACCGTCTCCTCA

276
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103265B04
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCCCTAGTTTCATCTTCAGCAACAATTACAT

GGAGTGGTACCGGCAGGCTCCAGGGAAGCAGCG

CGACTGGGTCGCACGTATTACAGGTCGCGGTAAC

ACAAACTATCTGGACTCCGTGAAGGGCCGATTCA

CCATCTCCAGAGACGACGCCAAGAATACGGTGT

ATCTAGAAATCGACAGCCTGAAACCTGAGGACA

CGGCCGTCTATTACTGTAGTGCACTCTGGTACGG

CGGGCGCGCATGGGGCAAAGGGACCCTGGTCAC

CGTCTCCTCA

277
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103275B05
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCTAGAGACAACGCCAAACGGGTGTATC

TGCAAATGAACAGCCTGACACCTGAGGACACGG

CCGTCTATTATTGTAATGCACTGCTACAACCGTC

GATTTATGACATTAGTCGCACATATTGGGGCCAG

GGGACCCTGGTCACCGTCTCCTCA

278
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTG

encoding F0103362B08
GTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTG

CAGCCTCTGTACGTCCCTTCAGTACCTCAGCCAT

GGGCTGGTTCCGCCAGGCTCCAGAGAAGGAGCG

TGAGGCTGTAGCAGGTATTCTGTGGAATGGTATT

GTCACATACTATGCAGACTCCGTGAAGGGCCGAT

TCACCATCTCCAGAGACAACGCCAAGAATGAAG

TATATCTGCAAATGAACAAACTGAAACCCGAGG

ACACGGCCGTTTATTATTGTGCATTAGATAGAGA

TTATGGTGGGCGATCTTTTTCGGCATATGAATAT

GAGTACTGGGGCCAGGGGACCCTGGTCACCGTCT

CCTCA

279
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103387G04
GCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGACCCGTCTTCAATATCAACAAGAT

GGCCTGGTACCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTGTCACCCCTACTGGTAGT

ATAAGTTATACTGACTCCGTGAAGGGCCGATTCA

CCATTTCTAGAGACGGCTCCAAGCGGTGGTCTCT

ACAAATGAACAGCCTGACACCTGAGGACACGGC

CGTCTATTACTGTAACGCTTTACTACAACCGGAT

AGTTATTCTAATACGCGCACATATTGGGGCCAGG

GGACCCTGGTCACCGTCTCCTCA

280
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103387G05
GTGCAGGCTGGGGGGTCACTGAGACTCTCCTGTG

ACGCCTCTGGAAGGATCCTCCGTATCGGCTACAT

GAGGTGGCACCGCCAGGGTGCAGGGAAGCAGCG

CGAGTTTGTCGCGCGTATTACTGATGATAGTGCT

ACAGACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAGAACACGGTG

TATCTGCAAATGAACAACCTGAATCCTGAGGACA

CGGCCGTCTATTATTGTGAGGCGTTGGTGACTGC

GAGTGTACGTGGTGGGAGTATACATTCTGGTACC

TATTGGGGCCGGGGGACCCTGGTCACCGTCTCCT

CA

281
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTG

encoding F0103454D07
GTGCAGCCTGGAGGATCACTTAGACTGTCCTGTG

TAGCCTCCGGGGGCATCATCAATATCAATTACAT

TGCCTGGTACCGCCAGACTCCAGGGAAGCAGCG

CGACTTGGTCGCTCGTATTAGTAGTGATGATACA

ATAAAGTATGGCGACTCCGTGAAGGGCCGATTC

GCCATGTCCAGAGACAAGGTCAAGAATATGGTG

CATCTACAAATGAACAGCCTGACTACCGAGGAC

ACAGGTGTCTATGTCTGTTCAGCCCTCATCACGC

CTTGGACAGGAGACACCCGGACCTATTGGGGCC

GGGGGACCCTGGTCACCGTCTCCTCA

282
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTG

encoding F0103464B09
GTGCAGCCTGGAGGATCACTAAGACTGTCCTGTG

CAACAACCTCTAGAGCTTTCATCAGGGACGTTTT

CACGGGCTGGTATCGCCGGGTTCCCGGGAAGGA

GCGCGAATTGGTCGCTCGCATTTACAATGGCGGT

AACACAAATTATGCAGACTTCGCGAAGGGCCGA

TTCTCCATCTCCAGGGACAACGCCAAGAAGATGG

TGACTCTGAGAATGAGCAATCTGAAACCTGAGG

ACACAGGGGTCTATTACTGCCTTTTTTCGGGTAC

AATCAATACTGGCAGAGAGTATCGGTCTGGAGA

CTACTGGGGCCAGGGGACCCTGGTCACCGTCTCC

TCA

283
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTG

encoding F0103464B09
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACCGCAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATATAGCACCAATGGTGGTGAT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCAGCAGAGACAACGCCAAACGGGTGTAT

CTGCAAATGAACAGCCTGACACCTGAGGACACG

GCCGTCTATTATTGTCGCGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

284
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301635
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTAATGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

285
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301636
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTAATGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

286
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301637
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTAATGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

287
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301638
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTAATGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

288
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301639
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGACCGTG

TATCTGCAAATGAACAGCCTGCGCCCTGAGGACA

CGGCCCTGTATTATTGTAATGCACTGCTACAACC

GTCGATTTATGACATTAGTCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

289
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301640
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGACCGTG

TATCTGCAAATGAACAGCCTGCGCCCTGAGGACA

CGGCCCTGTATTATTGTAATGCACTGCTACAACC

GTCGATTTATGACATTAGTCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

290
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301641
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACACCGTG

TATCTGCAAATGAACAGCCTGCGCCCTGAGGACA

CGGCCCTGTATTATTGTAATGCACTGCTACAACC

GTCGATTTATGACATTAGTCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

291
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301642
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACAGTAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCAAGTAGCACCAATGGTGGTAGT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACACCGTG

TATCTGCAAATGAACAGCCTGCGCCCTGAGGACA

CGGCCCTGTATTATTGTAATGCACTGCTACAACC

GTCGATTTATGACATTAGTCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

292
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301652
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACCGCAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATATAGCACCAATGGTGGTGAT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTCGCGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

293
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301653
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACCGCAT

GGCCTGGTATCGCCGGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATATAGCACCAATGGTGGTGAT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTCGCGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

294
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301654
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACCGCAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATATAGCACCAATGGTGGTGAT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAACGGGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTCGCGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

295
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTT

encoding F010301655
GTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGAAGCATCTTCAATATCAACCGCAT

GGCCTGGTATCGCCAGGCTCCAGGGAAGCAGCG

CGAATTGGTCGCATATAGCACCAATGGTGGTGAT

ACAAACTATGCAGACTCCGTGAAGGGCCGATTC

ACCATCTCCAGAGACAACGCCAAAAACGTGTAT

CTGCAAATGAACAGCCTGCGCCCTGAGGACACG

GCCCTGTATTATTGTCGCGCACTGCTACAACCGT

CGATTTATGACATTAGTCGCACATATTGGGGCCA

GGGGACCCTGGTCACCGTCTCCTCA

296
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGTGC

encoding F010301556
AGGCTGGGGGGTCACTGAGACTCTCCTGTGCTGCCTC

TGGAAGAATCCTCCGTATCGGCTACATGAGGTGGCAC

CGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGCG

CGTATTACTGGTGGTAGTGCTACAGGCTATGCAGACT

CCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC

CAAGAACACGGTGTATCTGCAAATGAACAACCTGAAT

CCTGAGGACACGGCCGTCTATTATTGTGAGGCGTTGG

TGACTGCGAGTGTACGTGGTGGGAGTATACATTCTGG

AACCTATTGGGGCCGGGGGACCCTGGTCACCGTCTCC

TCA

297
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGTGC

encoding F010301563
AGGCTGGGGGGTCACTGAGACTCTCCTGTGCTGCCTC

TGGAAGAATCCTCCGTATCGGCTACATGAGGTGGCAC

CGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGCG

CGTATTACTGATGATAGTGCTACAGGCTATGCAGACT

CCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC

CAAGAACACGGTGTATCTGCAAATGAACAACCTGAAT

CCTGAGGACACGGCCGTCTATTATTGTGAGGCGTTGG

TGACTGCGAGTGTACGTGGTGGGAGTATACATTCTGG

AACCTATTGGGGCCGGGGGACCCTGGTCACCGTCTCC

TCA

298
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301849
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

299
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTCGTG

encoding F010301850
CAGCCGGGGGGGTCACTGAGACTCTCCTGTGCGGCCA

GCGGAAGGATTCTCCGTATCGGCTACATGAGGTGGTA

TCGCCAGGCGCCGGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGGTTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCG

CCCTGAGGACACGGCCCTGTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GCACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

300
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301643
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGCCGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

301
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301644
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGTA

TCGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

302
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301645
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGCTGCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

303
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301646
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGGTCCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

304
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301647
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGGTGCAGGGAAGCAGCGCGAGCTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

305
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTG

encoding F010301648
CAGCCTGGGGGGTCACTGAGACTCTCCTGTGATGCCT

CTGGAAGGATACTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGGTGCAGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGACTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACTCCCTGCG

ACCTGAGGACACGGCCCTCTATTATTGTAACGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GAACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

306
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTCGTG

encoding F010301649
CAGCCGGGGGGGTCACTGAGACTCTCCTGTGCGGCCA

GCGGAAGGATTCTCCGTATCGGCTACATGAGGTGGCA

CCGCCAGGCGCCGGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGGTTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG

CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCG

CCCTGAGGACACGGCCCTGTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GCACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

307
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTCGTG

encoding F010302307
CAGCCGGGGGGGTCACTGAGACTCTCCTGTGCGGCCA

GCGGAAGGATTCTCCGTATCGGCTACATGAGGTGGTA

TCGCCAGGCGCCGGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGGTTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACGCGG

CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCG

CCCTGAGGACACGGCCCTGTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GCACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

308
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTCGTG

encoding F010302308
CAGCCGGGGGGGTCACTGAGACTCTCCTGTGCGGCCA

GCGGAAGGATTCTCCGTATCGGCTACATGAGGTGGTA

TCGCCAGGCGCCGGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGGTTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACTATG

CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCG

CCCTGAGGACACGGCCCTGTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GCACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

309
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTCGTG

encoding F010302309
CAGCCGGGGGGGTCACTGAGACTCTCCTGTGCGGCCA

GCGGAAGGATTCTCCGTATCGGCTACATGAGGTGGTA

TCGCCAGGCGCCGGGGAAGCAGCGCGAGTTTGTCGC

GCGTATTACTGATGATAGTGCTACAGGTTATGCAGAC

TCCGTGAAGGGCCGATTCACCATCTCCAGAGACCAGG

CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGCG

CCCTGAGGACACGGCCCTGTATTATTGTGAGGCGTTG

GTGACTGCGAGTGTACGTGGTGGGAGTATACATTCTG

GCACCTATTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

310
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGTGGTGGAGTTGTTC

encoding F010302391
AACCCGGTGGTTCTTTGAGATTGTCTTGCGCCGCTTCC

GGTAGAATCTTGCGTATCGGTTACATGCGTTGGTATA

GACAAGCTCCCGGTAAGCAAAGAGAGTTCGTCGCCA

GAATCACCGGAGGTTCTGCTACTGGTTATGCTGATTC

CGTCAAGGGAAGATTTACCATCTCCAGAGACAACGCT

AAGAACACTGTTTATTTGCAAATGAACTCCTTGAGAC

CCGAAGATACCGCTTTGTACTACTGCGAGGCTTTGGT

CACTGCTTCCGTTAGAGGAGGATCTATCCACTCCGGT

ACTTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCT

CA

311
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGTGGTGGTGTTGTTC

encoding F010302392
AGCCCGGTGGTTCCTTGAGATTGTCTTGTGCTGCTTCC

GGTAGAATCTTGAGAATCGGTTACATGAGATGGTACA

GACAAGCCCCCGGTAAGCAGAGAGAGTTCGTCGCCA

GAATCACTGGAGGATCTGCTACTGGTTACGCTGACTC

CGTCAAGGGAAGATTCACCATCTCCAGAGATCAAGCT

AAGAACACCGTCTACTTGCAGATGAACTCCTTGAGAC

CAGAGGACACCGCTTTGTACTACTGTGAGGCTTTAGT

TACTGCTTCCGTTAGAGGTGGTTCCATTCACTCTGGTA

CTTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTC

A

312
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301868
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

313
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301869
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAAACCGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

314
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301870
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCAACTTTT

CGGGTACAATCAATACTGGCAGAGAGTATCGGTCTGG

AGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCC

TCA

315
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301871
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

316
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301872
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

GCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

317
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301873
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

318
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301874
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGGAGCGCGAATTGGT

CGCTCGCATTTACAATGGCGGTAACACAAATTATGCA

GACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACA

ACGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCT

GCGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTT

CGGGTACAATCAATACTGGCAGAGAGTATCGGTCTGG

AGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCC

TCA

319
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301875
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACAGCGCGAAGGGCCGATTCACCATCTCCAGGGACA

ACGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCT

GCGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTT

CGGGTACAATCAATACTGGCAGAGAGTATCGGTCTGG

AGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCC

TCA

320
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301876
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGTGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

321
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301877
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAACATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

322
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTGGTG

encoding F010301892
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCTAGAGCTTTCATCAGGGACCTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAGCAATCT

GAAACCTGAGGACACAGGGGTCTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

323
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010301893
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGCAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGCAAATGAACAGCCTG

CGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

324
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301932
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAGCAATCT

GCGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

325
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301933
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCACCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAGCAATCT

GCGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

326
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301934
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGACCGTGACTCTGAGAATGAGCAATCT

GCGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

327
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301935
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGTATCTGAGAATGAGCAATCTG

CGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

328
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301936
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGCAAATGAGCAATCTG

CGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTTTC

GGGTACAATCAATACTGGCAGAGAGTATCGGTCTGGA

GACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

CA

329
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301937
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAACAATCT

GCGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

330
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301938
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAGCAGCCT

GCGCCCTGAGGACACAGGGCTGTATTACTGCCTTTTT

TCGGGTACAATCAATACTGGCAGAGAGTATCGGTCTG

GAGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC

CTCA

331
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTTGTG

encoding F010301939
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAACAA

CCTCCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGTTCCCGGGAAGGAGCGCGAATTGGTC

GCTCGCATTTACAATGGCGGTAACACAAATTATGCAG

ACTTCGCGAAGGGCCGATTCTCCATCTCCAGGGACAA

CGCCAAGAAGATGGTGACTCTGAGAATGAGCAATCT

GCGCCCTGAGGACACAGCGCTGTATTACTGCCTTTTTT

CGGGTACAATCAATACTGGCAGAGAGTATCGGTCTGG

AGACTACTGGGGCCAGGGGACCCTGGTCACCGTCTCC

TCA

332
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302333
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

333
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302334
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

334
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302335
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

335
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302336
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

336
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302337
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

337
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302338
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

338
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302339
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

339
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302340
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACCTGTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

340
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302341
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

341
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302342
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

342
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302343
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

343
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302344
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

344
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302345
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

345
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302346
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

346
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302347
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

347
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302348
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACCTGTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

348
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302349
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACAAATTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

349
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302350
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

350
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302351
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

351
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302352
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

352
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302353
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

353
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302354
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

354
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302355
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

355
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302356
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACCTGTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACAAATTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

356
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302357
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACAAATTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

357
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302358
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

358
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302359
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

359
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302360
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

360
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302361
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

361
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302362
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

362
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302363
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAGGCGGTAACACACAGTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

363
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302364
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCCATAGAGCTTTCATCAGGGACCTGTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACGAAAGCGGTAACACAAATTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

364
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302365
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGGCGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

365
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302366
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGGCGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

366
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302367
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGAGCTTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCAGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGGCGGTAACACAAATTATGCA

GACAGCGTGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

367
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGAGTGGTG

encoding F010302368
CAGCCTGGAGGATCACTAAGACTGTCCTGTGCAGCGA

GCAGCAGACAGTTCATCAGGGACGTTTTCACGGGCTG

GTATCGCCGGGCGCCCGGGAAGCAGCGCGAATTGGT

CGCTCGCATTTACAATGAAGGTAACACAAATTATGCA

GACAGCGCGAAGGGCCGATTCACCATCTCCAGGGAC

AACGCCAAGAAAACCGTGTATCTGCAAATGAACAGC

CTGCGCCCTGAGGACACAGCGCTGTATTACTGCCTTT

TTTCGGGTACAATCAGCACTGGCAGAGAGTATCGGTC

TGGAGACTACTGGGGCCAGGGGACCCTGGTCACCGTC

TCCTCA

368
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGCG

encoding F010301656
CAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCT

CTGGACCCGTCTTTAATATCAACCGCATGGCCTGGTA

TCGCCGGGCTCCAGGGAAGCAGCGCGAATTGGTCGC

ATATGTCACCCCTACTGGTGATATAAGTTATACTGAC

TCCGTGAAGGGCCGATTCACCATTTCTAGGGACGGCT

CCAAGCGGTGGTCTCTACAAATGAACAGCCTGACACC

TGAGGACACGGCCGTCTATTACTGTCGCGCTTTACTA

CAACCGGATAGTTATTCTAATACGCGCACATATTGGG

GCCAGGGGACCCTGGTCACCGTCTCCTCA

369
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301840
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTCC

AAGCGGACCTGGTCTCTACAAATGAACAGCCTGCGCC

CTGAGGACACGGCCCTGTATTACTGTCGCGCTTTACT

ACAACCGGATAGTTATTCTAATACGCGCACATATTGG

GGCCAGGGGACCCTGGTCACCGTCTCCTCA

370
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301841
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTCC

AAGCGGTGGTCTCTACAAATGAACAGCCTGCGCCCTG

AGGACACGGCCCTGTATTACTGTCGCGCTTTACTACA

ACCGGATAGTTATTCTAATACGCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

371
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301842
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGTGGTCTCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

372
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301843
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACAACTCC

AAGCGGTGGTCTCTACAAATGAACAGCCTGCGCCCTG

AGGACACGGCCCTGTATTACTGTCGCGCTTTACTACA

ACCGGATAGTTATTCTAATACGCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

373
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301844
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTCC

AAGAACTGGTCTCTACAAATGAACAGCCTGCGCCCTG

AGGACACGGCCCTGTATTACTGTCGCGCTTTACTACA

ACCGGATAGTTATTCTAATACGCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

374
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301845
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTCC

AAGCGGGTCTCTCTACAAATGAACAGCCTGCGCCCTG

AGGACACGGCCCTGTATTACTGTCGCGCTTTACTACA

ACCGGATAGTTATTCTAATACGCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

375
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301846
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATACTGACTC

CGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTCC

AAGCGGTGGTATCTACAAATGAACAGCCTGCGCCCTG

AGGACACGGCCCTGTATTACTGTCGCGCTTTACTACA

ACCGGATAGTTATTCTAATACGCGCACATATTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

376
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301847
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACAACTC

CAAGAACGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

377
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301848
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACAACTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

378
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301865
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

379
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010301866
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGAACGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

380
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302310
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

381
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302311
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

382
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302312
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

383
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302313
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCC

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

384
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302314
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

385
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302315
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

386
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302316
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

387
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302317
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

388
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302318
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

389
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302319
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

390
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302320
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

391
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302321
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

392
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302322
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

393
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302323
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

394
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302324
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

395
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302325
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

396
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302326
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

397
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302327
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

398
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302328
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCAGTTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

399
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302329
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

400
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302330
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGCGTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

401
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302331
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

402
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302332
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGCGCCGCTATTCTAATACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

403
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302370
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

404
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302371
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAACGTGTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

405
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302372
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGACCGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGGATGTGTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

406
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302383
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGGTGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAGCAGTTATTCTGGCACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

407
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302384
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCGGTGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

408
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302385
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCCAGGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAGCAGTTATTCTGGCACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

409
Nucleotide sequence
GAGGTGCAATTGGTGGAGTCTGGGGGAGGCGTGGTTC

encoding F010302386
AGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC

TGGACCCGTGTTCAATATCAACCGCATGGCCTGGTAT

CGCCAGGCTCCAGGGAAGCAGCGCGAATTGGTCGCA

TATGTCACCCCTACTGGTGATATAAGTTATGCGGACT

CCGTGAAGGGCCGATTCACCATTTCTCGCCAGGGCTC

CAAGCGGGTGTATCTACAAATGAACAGCCTGCGCCCT

GAGGACACGGCCCTGTATTACTGTCGCGCTTTACTAC

AACCGAGCAGTTATTCTATTACGCGCACATATTGGGG

CCAGGGGACCCTGGTCACCGTCTCCTCA

410
F0103240B04 (No tag)
EVQLVESGGGLVQAGGSLRLSCAASGGTGRRYAMGWF

RQAPGKEREIVAAIRWSAMTYYADDGKGRFTISRDNAK

NTVYLQMNSLKPEDTAIYYCAYTWDYFKYDQVRAYRG

WGQGTLVTVSS

411
F0103478E09 (No tag)
EVQLVESGGGLVQAGGSLRLSCAASGRAFSTLAMGWF

RQAPGKEREFVAAISRNGNNSATGDSLKGRFTISRDSTK

STVF

LQMNTLKPEDTAVYYCAAISTPSASHPYVRKESYRYWG

QGTLVTVSS

412
F0103492E09 (No tag)
EVQLVESGGGLVQAGGSLRLSCAASKSILSFAYMRWYR

QAPGKQREFVASIAIGGATSYTDSVKGRFTISRDNAKNT

VYLQMNSLKPEDTAVYYCSAPAGQYRGQGTLVTVSS

413
F0103500E03 (No tag)
EVQLVESGGGLVQPGGSLRLSCAASGRTFSRYQMGWFR

QAPGKEREFVAYISWSGSTRYVDSVKGRFTISRDNAKNT

VYLQMNSLKPEDTAVYHCAAGTAGIISSRPETYDSWGQ

GTLVTVSS

414
F0103505D08 (No tag)
EVQLVESGGGLVQAGGSLRLSCVTSGRTSDLSTMNWFR

QAPGKEREFVARITRRGSTYYAESVKERFIISRDNAKNT

VYL

QMNSLKPEDTANYYCTAASEMGYHYRGQGTLVTVSS

415
F0103495F09 (No tag)
EVQLVESGGGLVQAGSSLSLSCAASGRALSTYAMGWFR

QAPGKEREFVARISRSGITTYYTDSVKGRFTISRDRAKDT

VY

LQMNSLKPEDTAIYLCAADASTNPAGYYLRNRYDYWG

QGTLVTVSS

416
F0103240B04-CDR1
GGTGRRYAMGW

417
F0103240B04-CDR2
AIRWSAMTY

418
F0103240B04-CDR3
TWDYFKYDQVRAYRG

419
F0103478E09-CDR1
GRAFSTLAMG

420
F0103478E09-CDR2
ISRNGNNS

421
F0103478E09-CDR3
ISTPSASHPYVRKESYRY

422
F0103492E09-CDR1
KSILSFAYMR

423
F0103492E09-CDR2
SIAIGGATS

424
F0103492E09-CDR3
PAGQYR

425
F0103500E03-CDR1
GRTFSRYQMG

426
F0103500E03-CDR2
YISWSGSTR

427
F0103500E03-CDR3
GTAGIISSRPETYDS

428
F0103505D08-CDR1
GRTSDLSTMN

429
F0103505D08-CDR2
RITRRGSTY

430
F0103505D08-CDR3
ASEMGYHYR

431
F0103495F09-CDR1
GRALSTYAMG

432
F0103495F09-CDR2
RISRSGITT

433
F0103495F09-CDR3
DASTNPAGYYLRNRYDY

434
F103275B05(N93R)
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

435
F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

(E1D, N93R)
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

436
F0103478E09 (L108Q)
EVQLVESGGGLVQAGGSLRLSCAASGRAFSTLAMGWF

RQAPGKEREFVAAISRNGNNSATGDSLKGRFTISRDSTK

STVFLQMNTLKPEDTAVYYCAAISTPSASHPYVRKESYR

YWGQGTQVTVSS

437
F0103505D08 (L108Q)
EVQLVESGGGLVQAGGSLRLSCVTSGRTSDLSTMNWFR

QAPGKEREFVARITRRGSTYYAESVKERFIISRDNAKNT

VYLQMNSLKPEDTANYYCTAASEMGYHYRGQGTQVT

VSS

438
F0103500E03
EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYQMGWF

(P14A, L108Q)
RQAPGKEREFVAYISWSGSTRYVDSVKGRFTISRDNAK

NTVYLQMNSLKPEDTAVYHCAAGTAGIISSRPETYDSW

GQGTQVTVSS

439
F010302375:F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

(E1D, N93R)-50GS-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

F0103478E09(L108Q)-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

FLAG3-HIS6
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASGRAFSTLAMGWFRQAPGKEREFVAAISRNGN

NSATGDSLKGRFTISRDSTKSTVFLQMNTLKPEDTAVYY

CAAISTPSASHPYVRKESYRYWGQGTQVTVSSGAADYK

DHDGDYKDHDIDYKDDDDKGAAHHHHHH

440
F010302377:F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

(E1D, N93R)-50GS-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

F0103492E09-FLAG3-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

HIS6
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASKSILSFAYMRWYRQAPGKQREFVASIAIGGAT

SYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

SAPAGQYRGQGTLVTVSSGAADYKDHDGDYKDHDIDY

KDDDDKGAAHHHHHH

441
F010302378:F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

(E1D,N93R)-50GS-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

F0103495F09-FLAG3-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

HIS6
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGSSL

SLSCAASGRALSTYAMGWFRQAPGKEREFVARISRSGIT

TYYTDSVKGRFTISRDRAKDTVYLQMNSLKPEDTAIYLC

AADASTNPAGYYLRNRYDYWGQGTLVTVSSGAADYK

DHDGDYKDHDIDYKDDDDKGAAHHHHHH

442
F010302379:F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

(E1D, N93R)-50GS-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

F0103500E03(P14A, L108Q)-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

FLAG3-HIS6
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASGRTFSRYQMGWFRQAPGKEREFVAYISWSGS

TRYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYH

CAAGTAGIISSRPETYDSWGQGTQVTVSSGAADYKDHD

GDYKDHDIDYKDDDDKGAAHHHHHH

443
F010302380:F0103275B05
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

(E1D, N93R)-50GS-
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

F0103505D08(L108Q)-
RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

FLAG3-HIS6
SRTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG

SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ

LVESGGGLVQAGGSLRLSCVTSGRTSDLSTMNWFR

QAPGKEREFVARITRRGSTYYAESVKERFIISRDNA

KNTVYLQMNSLKPEDTANYYCTAASEMGYHYRG

QGTQVTVSSGAADYKDHDGDYKDHDIDYKDDDD

KGAAHHHHHH

444
F010300191:F0103275B05-
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

50GS-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

F0103240B04-FLAG3-
VYLQMNSLTPEDTAVYYCNALLQPSIYDISRTYWGQGT

HIS6
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASGGTGRRYAMGWFRQAPGKEREIVAAIRWSA

MTYYADDGKGRFTISRDNAKNTVYLQMNSLKPEDTAIY

YCAYTWDYFKYDQVRAYRGWGQGTLVTVSSGAADYK

DHDGDYKDHDIDYKDDDDKGAAHHHHHH

445
F010302375 (No Tag):
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

F0103275B05(E1D, N93R)-
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

50GS-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

F0103478E09(L108Q)
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASGRAFSTLAMGWFRQAPGKEREFVAAISRNGN

NSATGDSLKGRFTISRDSTKSTVFLQMNTLKPEDTAVYY

CAAISTPSASHPYVRKESYRYWGQGTQVTVSS

446
F010302377 (No
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

Tag):F0103275B05
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

(E1D, N93R)-50GS-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

F0103492E09
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASKSILSFAYMRWYRQAPGKQREFVASIAIGGAT

SYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

SAPAGQYRGQGTLVTVSS

447
F010302378 (No
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

Tag):F0103275B05
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

(E1D, 93R)-50GS-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

F0103495F09
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGSSL

SLSCAASGRALSTYAMGWFRQAPGKEREFVARISRSGIT

TYYTDSVKGRFTISRDRAKDTVYLQMNSLKPEDTAIYLC

AADASTNPAGYYLRNRYDYWGQGTLVTVSS

448
F010302379 (No
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMAWYR

Tag):F0103275B05
RAPGKQRELVASSTNGGSTNYADSVKGRFTISRDNAKR

(E1D, N93R)-50GS-
VYLQMNSLTPEDTAVYYCRALLQPSIYDISRTYWGQGT

F0103500E03(P14A, L108Q)
LVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG

GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL

RLSCAASGRTFSRYQMGWFRQAPGKEREFVAYISWSGS

TRYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYH

CAAGTAGIISSRPETYDSWGQGTQVTVSS

449
F010302380 (No
DVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

Tag):F0103275B05
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

(E1D, N93R)-50GS-
RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

F0103505D08(L108Q)
SRTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG

SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ

LVESGGGLVQAGGSLRLSCVTSGRTSDLSTMNWFR

QAPGKEREFVARITRRGSTYYAESVKERFIISRDNA

KNTVYLQMNSLKPEDTANYYCTAASEMGYHYRG

QGTQVTVSS

450
F010300191 (No
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

Tag):F0103275B05-
WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

50GS-F0103240B04
RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRTYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG

SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ

LVESGGGLVQAGGSLRLSCAASGGTGRRYAMGWF

RQAPGKEREIVAAIRWSAMTYYADDGKGRFTISRD

NAKNTVYLQMNSLKPEDTAIYYCAYTWDYFKYDQ

VRAYRGWGQGTLVTVSS

451
F0103PMP478E09
EVQLVESGGGLVQAGGSLRLSCAASGRAFSTLAM

GWFRQAPGKEREFVAAISRNGNNSATGDSLKGRFT

ISRDSTKSTVFLQMNTLKPEDTAVYYCAAISTPSAS

HPYVRKESYRYWGQGTQVTVSSAAADYKDHDGD

YKDHDIDYKDDDDKGAAHHHHHHKAAGGGGG

452
F0103PMP492E09
EVQLVESGGGLVQAGGSLRLSCAASKSILSFAYMRWYR

QAPGKQREFVASIAIGGATSYTDSVKGRFTISRDNAKNT

VYLQMNSLKPEDTAVYYCSAPAGQYRGQGTLVTVSSA

AADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHHKA

AGGGGG

453
F0103PMP495F09
EVQLVESGGGLVQAGSSLSLSCAASGRALSTYAMGWFR

QAPGKEREFVARISRSGITTYYTDSVKGRFTISRDRAKDT

VY

LQMNSLKPEDTAIYLCAADASTNPAGYYLRNRYDYWG

QGTLVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGA

AHHHHHHKAAGGGGG

454
F0103PMP500E03
EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYQMGWF

RQAPGKEREFVAYISWSGSTRYVDSVKGRFTISRDNAK

NTVYLQMNSLKPEDTAVYHCAAGTAGIISSRPETYDSW

GQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKG

AAHHHHHHKAA

455
F0103PMP505D08
EVQLVESGGGLVQAGGSLRLSCVTSGRTSDLSTMNWFR

QAPGKEREFVARITRRGSTYYAESVKERFIISRDNAKNT

VYL

QMNSLKPEDTANYYCTAASEMGYHYRGQGTQVTVSSA

AADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHHKA

AGGGGG

456
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103240B04
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGG

CTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGTACA

GGCAGGAGATATGCCATGGGCTGGTTCCGCCAGGCTC

CAGGGAAGGAGCGTGAAATTGTAGCAGCGATTAGGT

GGAGTGCTATGACATACTATGCAGACGACGGGAAGG

GCCGATTCACCATCTCCAGAGACAACGCCAAGAACAC

GGTGTATCTCCAAATGAACAGCCTGAAACCTGAGGAC

ACGGCCATTTATTACTGTGCATACACTTGGGACTATTT

CAAGTATGACCAAGTCCGAGCGTATCGCGGCTGGGGC

CAGGGGACCCTGGTCACCGTCTCCTCA

457
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103478E09
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGG

GTCTCTGAGACTCTCCTGTGCTGCCTCTGGACGCGCCT

TCAGTACCTTGGCCATGGGCTGGTTCCGCCAGGCTCC

AGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGCCG

GAATGGTAATAACTCAGCCACTGGAGACTCCCTGAAG

GGCCGATTCACCATCTCCAGAGACAGCACCAAGAGC

ACGGTTTTTCTGCAAATGAATACGCTGAAACCTGAGG

ACACGGCCGTATATTACTGTGCAGCCATCTCGACACC

GTCCGCCAGTCATCCATACGTTCGCAAGGAAAGTTAT

AGATACTGGGGCCAGGGTACCCTGGTCACCGTCTCCT

CA

458
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103492E09
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGG

ATCTCTGAGACTCTCCTGTGCAGCCTCTAAAAGCATC

TTAAGTTTCGCTTACATGCGCTGGTACCGCCAGGCTC

CAGGGAAGCAGCGCGAGTTCGTCGCAAGTATTGCTAT

TGGAGGTGCCACAAGCTATACAGACTCCGTGAAGGG

CCGATTCACCATCTCCAGAGACAACGCCAAGAACACG

GTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACA

CAGCCGTCTATTACTGTAGTGCACCAGCCGGACAGTA

TCGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA

459
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103500E03
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGATTGGTGCAGCCGGGGGG

CTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCT

TCTCGCGCTATCAGATGGGCTGGTTCCGCCAGGCTCC

AGGGAAGGAGCGTGAGTTTGTAGCATATATTAGCTGG

AGTGGTAGTACACGTTATGTTGACTCCGTGAAGGGCC

GATTCACCATCTCCAGAGACAACGCCAAGAACACGGT

GTATCTGCAAATGAACAGCCTGAAACCTGAGGACAC

GGCCGTTTATCACTGTGCAGCAGGGACGGCCGGCATA

ATATCTAGTAGGCCTGAAACTTATGACTCATGGGGCC

AGGGGACCCTGGTCACCGTCTCCTCA

460
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103505D08
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGG

CTCTCTGAGACTCTCCTGTGTAACCTCTGGACGCACCT

CCGATTTGTCTACCATGAACTGGTTCCGCCAGGCTCC

AGGAAAGGAGCGTGAGTTTGTCGCACGCATCACTCGG

CGTGGTAGCACATACTATGCAGAGTCCGTGAAGGAAC

GATTCATCATCTCCAGAGACAACGCCAAGAACACGGT

GTATTTGCAAATGAACAGCCTGAAACCAGAGGACAC

GGCCAATTATTACTGTACTGCAGCCTCAGAAATGGGA

TATCACTACAGGGGCCAGGGGACCCTGGTCACCGTCT

CCTCA

461
Nucleotide sequence
ATGAAAAAGACCGCTATCGCGATTGCAGTGGCACTGG

encoding F0103495F09
CTGGTTTGGCCACCGTGGCCCAGGCCGAGGTGCAATT

(No tag)
GGTGGAGTCTGGGGGAGGTTTGGTGCAGGCTGGAAG

CTCTCTGAGTCTCTCCTGTGCAGCCTCTGGACGCGCCT

TGAGTACATACGCCATGGGCTGGTTCCGCCAGGCTCC

AGGGAAGGAGCGTGAGTTTGTAGCACGTATTAGCCG

GAGCGGGATTACAACATACTATACAGACTCCGTGAAG

GGCCGATTCACCATCTCCAGAGACCGCGCCAAGGACA

CGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGA

CACGGCCATTTATTTGTGTGCAGCAGACGCCTCAACC

AATCCTGCTGGATACTACCTTCGGAATCGTTATGACT

ACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA

462
50GS linker
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG

GSGGGGSGGGGSGGGGS

463
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding F010302375
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGATTTATGACATTAGTCG

CACATATTGGGGCCAGGGGACCCTGGTCACGGTC

TCCTCCGGAGGTGGTGGCAGCGGTGGAGGTGGTT

CTGGGGGTGGCGGTAGTGGCGGTGGTGGCTCAG

GTGGCGGTGGGTCAGGCGGTGGTGGCAGTGGTG

GGGGTGGCAGCGGTGGCGGTGGATCTGGTGGAG

GTGGTTCTGGAGGTGGAGGATCCGAGGTGCAGTT

GGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGG

GGGGTCTCTGAGACTCTCCTGTGCTGCCTCTGGA

CGCGCCTTCAGTACCTTGGCCATGGGCTGGTTCC

GCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAG

CAGCTATTAGCCGGAATGGTAATAACTCAGCCAC

TGGAGACTCCCTGAAGGGCCGATTCACCATCTCC

AGAGACAGCACCAAGAGCACGGTTTTTCTGCAA

ATGAATACGCTGAAACCTGAGGACACGGCCGTA

TATTACTGTGCAGCCATCTCGACACCGTCCGCCA

GTCATCCATACGTTCGCAAGGAAAGTTATAGATA

CTGGGGCCAGGGTACCCAGGTCACCGTCTCCTCA

464
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding F010302377
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGAT

TTATGACATTAGTCGCACATATTGGGGCCAGGGG

ACCCTGGTCACGGTCTCCTCCGGAGGTGGTGGCA

GCGGTGGAGGTGGTTCTGGGGGTGGCGGTAGTG

GCGGTGGTGGCTCAGGTGGCGGTGGGTCAGGCG

GTGGTGGCAGTGGTGGGGGTGGCAGCGGTGGCG

GTGGATCTGGTGGAGGTGGTTCTGGAGGTGGAG

GATCCGAGGTGCAGTTGGTGGAGTCTGGGGGAG

GCTTGGTGCAGGCTGGGGGATCTCTGAGACTCTC

CTGTGCAGCCTCTAAAAGCATCTTAAGTTTCGCT

TACATGCGCTGGTACCGCCAGGCTCCAGGGAAG

CAGCGCGAGTTCGTCGCAAGTATTGCTATTGGAG

GTGCCACAAGCTATACAGACTCCGTGAAGGGCC

GATTCACCATCTCCAGAGACAACGCCAAGAACA

CGGTGTATCTGCAAATGAACAGCCTGAAACCTGA

GGACACAGCCGTCTATTACTGTAGTGCACCAGCC

GGACAGTATCGGGGCCAGGGGACCCTGGTCACC

GTCTCCTCA

465
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding F010302378
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGAT

TTATGACATTAGTCGCACATATTGGGGCCAGGGG

ACCCTGGTCACGGTCTCCTCCGGAGGTGGTGGCA

GCGGTGGAGGTGGTTCTGGGGGTGGCGGTAGTG

GCGGTGGTGGCTCAGGTGGCGGTGGGTCAGGCG

GTGGTGGCAGTGGTGGGGGTGGCAGCGGTGGCG

GTGGATCTGGTGGAGGTGGTTCTGGAGGTGGAG

GATCCGAGGTGCAGTTGGTGGAGTCTGGGGGAG

GTTTGGTGCAGGCTGGAAGCTCTCTGAGTCTCTC

CTGTGCAGCCTCTGGACGCGCCTTGAGTACATAC

GCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAG

GAGCGTGAGTTTGTAGCACGTATTAGCCGGAGCG

GGATTACAACATACTATACAGACTCCGTGAAGG

GCCGATTCACCATCTCCAGAGACCGCGCCAAGG

ACACGGTGTATCTGCAAATGAACAGCCTGAAAC

CTGAGGACACGGCCATTTATTTGTGTGCAGCAGA

CGCCTCAACCAATCCTGCTGGATACTACCTTCGG

AATCGTTATGACTACTGGGGCCAGG

GGACCCTGGTCACCGTCTCCTCA

466
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding F010302379
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGAT

TTATGACATTAGTCGCACATATTGGGGCCAGGGG

ACCCTGGTCACGGTCTCCTCCGGAGGTGGTGGCA

GCGGTGGAGGTGGTTCTGGGGGTGGCGGTAGTG

GCGGTGGTGGCTCAGGTGGCGGTGGGTCAGGCG

GTGGTGGCAGTGGTGGGGGTGGCAGCGGTGGCG

GTGGATCTGGTGGAGGTGGTTCTGGAGGTGGAG

GATCCGAGGTGCAGTTGGTGGAGTCTGGGGGAG

GATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTC

CTGTGCAGCCTCTGGACGCACCTTCTCGAGGTAT

CAGATGGGCTGGTTCCGCCAGGCTCCAGGGAAG

GAGCGTGAGTTTGTAGCATATATTAGCTGGAGTG

GTAGTACACGTTATGTTGACTCCGTGAAGGGCCG

ATTCACCATCTCCAGAGACAACGCCAAGAACAC

GGTGTATCTGCAAATGAACAGCCTGAAACCTGA

GGACACGGCCGTTTATCACTGTGCAGCAGGGAC

GGCCGGCATAATATCTAGTAGGCCTGAAACTTAT

GACTCATGGGGCCAGGGGACCCAGG

TCACCGTCTCCTCA

467
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

F010302380(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGAT

TTATGACATTAGTCGCACATATTGGGGCCAGGGG

ACCCTGGTCACGGTCTCCTCCGGAGGTGGTGGCA

GCGGTGGAGGTGGTTCTGGGGGTGGCGGTAGTG

GCGGTGGTGGCTCAGGTGGCGGTGGGTCAGGCG

GTGGTGGCAGTGGTGGGGGTGGCAGCGGTGGCG

GTGGATCTGGTGGAGGTGGTTCTGGAGGTGGAG

GATCCGAGGTGCAGTTGGTGGAGTCTGGGGGAG

GATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTC

CTGTGTAACCTCTGGACGCACCTCCGATTTGTCT

ACCATGAACTGGTTCCGCCAGGCTCCAGGAAAG

GAGCGTGAGTTTGTCGCACGCATCACTCGGCGTG

GTAGCACATACTATGCAGAGTCCGTGAAGGAAC

GATTCATCATCTCCAGAGACAACGCCAAGAACA

CGGTGTATTTGCAAATGAACAGCCTGAAACCAG

AGGACACGGCCAATTATTACTGTACTGCAGCCTC

AGAAATGGGATATCACTACAGGGGCCAGGGGAC

CCAGGTCACCGTCTCCTCA

468
Nucleotide sequence
ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATT

encoding F010302391
CGCAGCATCCTCCGCATTAGCTGCTCCAGTCAAC

(No tag)
ACTACAACAGAAGATGAAACGGCACAAATTCCG

GCTGAAGCTGTCATCGGTTACTCAGATTTAGAAG

GGGATTTCGATGTTGCTGTTTTGCCATTTTCCAAC

AGCACAAATAACGGGTTATTGTTTATAAATACTA

CTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGT

ATCTCTCGAAAAGAGAGACGTGCAATTGGTGGA

GTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCT

CTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCT

TCAATATCAACAGTATGGCCTGGTATCGCCGGGC

TCCAGGGAAGCAGCGCGAATTGGTCGCAAGTAG

CACCAATGGTGGTAGTACAAACTATGCAGACTCC

GTGAAGGGCCGATTCACCATCTCTAGAGACAAC

GCCAAACGGGTGTATCTGCAAATGAACAGCCTG

ACACCTGAGGACACGGCCGTCTATTATTGTCGTG

CACTGCTACAACCGTCGAT

TTATGACATTAGTCGCACATATTGGGGCCAGGGG

ACCCTGGTCACGGTCTCCTCCGGAGGTGGTGGCA

GCGGTGGAGGTGGTTCTGGGGGTGGCGGTAGTG

GCGGTGGTGGCTCAGGTGGCGGTGGGTCAGGCG

GTGGTGGCAGTGGTGGGGGTGGCAGCGGTGGCG

GTGGATCTGGTGGAGGTGGTTCTGGAGGTGGAG

GATCCGAGGTGCAGTTGGTGGAGTCTGGGGGAG

GATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTC

CTGTGCAGCCTCTGGACGCACCGCTAGTATCTAT

GCCATGGCCTGGTTCCGCCAGGCTCAGGGGAAG

GAGCGTGAATTTGTCGCAGTTATTACCCGGAGTG

GTGGAACGATCGTCTATGCAGACTCCGTGAAGG

GCCGATTCACCATCTCCAGAGACGACGCCAAGA

ACACTGTGTGGTTGCAAATGAGCGCTCTGAGACC

TGAGGACACAGCCGTATATTTCTGTAATGCGGTT

GCGGTCGAAGACGGGATGAACGTTATGAATTATT

GGGGCCAGGGGACCCTGGTCACCG

TCTCCTCA

469
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

470
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(L234A L235A D265S)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT

CVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

471
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(L234A L235A P329G)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT

CVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK

472
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(L235E)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPELEGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

473
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(D265A)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

474
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(D265A N297G)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

475
Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(E233A/L235A)
PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

HTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK

476
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV

DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI

EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

477
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(D265S)
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV

SVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI

EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

478
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(P329G)
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV

DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLGAPI

EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

479
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(D265A)
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV

AVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI

EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

480
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(D265A N297G)
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV

AVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFGS

TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI

EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK

481
Human IgG2 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(V234A G237A P238S
PSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC

H268A V309L A330S
PPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVV

P331S X378S/A)(See
DVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

IgGsigma SEQ ID
TFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE

No: 78 in
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV

WO2017079112)
KGFYPSDIXVEWESNGQPENNYKTTPPMLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

KSLSLSPGK

482
Human IgG4 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(S228P)
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC

PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI

EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT

QKSLSLSLGK

483
Human IgG4 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(S228P P329G)
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC

PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSS

IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY

TQKSLSLSLGK

484
Human IgG4 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(S228P D265A)
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC

PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI

EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT

QKSLSLSLGK

485
Human IgG4 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

Constant domain
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

(S228P D265A N297G)
PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC

PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFG

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI

EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT

QKSLSLSLGK

486
F010301657
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAGILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYQGRSFSAKEYEYWGQGTLVTVSS

487
F010301658
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAAILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYQGRSFSAKEYEYWGQGTLVTVSS

488
F010301659
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAAILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYGGRSFSAYEYEYWGQGTLVTVSS

489
F010301661
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAAILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRDYGGRSFSAKEYEYWGQGTLVTVSS

490
F010301662
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAGILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYQGRSFSAYEYEYWGQGTLVTVSSA

491
F010301663
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAGILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYGGRSFSAKEYEYWGQGTLVTVSS

492
F010301664
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAGILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRDYQGRSFSAKEYEYWGQGTLVTVSS

493
F010301665
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAAILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRAYGGRSFSAKEYEYWGQGTLVTV

494
F010301666
MKKTAIAIAVALAGLATVAQAEVQLVESGGGLVQPGGS

LRLSCAASVRPFSTSAMGWFRQAPEKEREAVAAILWNG

IVTYYADSVKGRFTISRDNAKNEVYLQMNKLKPEDTAV

YYCALDRDYQGRSFSAKEYEYWGQGTLVTVSS

495
F010301867
EVQLVESGGGLVQPGGSLRLSCATTSRQFIRDVFTG

WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

496
F010301878
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNEGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

497
F010301879
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNQGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

498
F010301880
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNSGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

499
F010301881
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNWGNTNYADFAKGRFSI

SRDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

500
F010301882
EVQLVESGGGLVQPGGSLRLSCATTSRAFVRDVFT

GWYRRVPGKERELVARIYNGGNTNYADFAKGRFSI

SRDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

501
F010301883
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARVYNGGNTNYADFAKGRFSI

SRDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

502
F010301884
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYAGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

503
F010301885
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYEGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

504
F010301886
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNGGNTQYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

505
F010301887
EVQLVESGGGLVQPGGSLRLSCATTSRAFIQDVFTG

WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

506
F010301888
EVQLVESGGGLVQPGGSLRLSCATTHRAFIRDVFT

GWYRRVPGKERELVARIYNGGNTNYADFAKGRFSI

SRDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

507
F010301889
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFAGTINTG

REYRSGDYWGQGTLVTVSS

508
F010301890
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFVG

WYRRVPGKERELVARIYNGGNTNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

509
F010301891
EVQLVESGGGLVQPGGSLRLSCATTSRAFIRDVFTG

WYRRVPGKERELVARIYNGGNVNYADFAKGRFSIS

RDNAKKMVTLRMSNLKPEDTGVYYCLFSGTINTG

REYRSGDYWGQGTLVTVSS

510
F010300534
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGYVNYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

511
F010300535
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFWGGYTNYVDSVKGRFT

ISRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQ

GTLVTVSS

512
F010300536
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGYTTYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

513
F010301055
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFWGGYVNYVDSVKGRFT

ISRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQ

GTLVTVSS

514
F010301059
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGYVNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

515
F010301080
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFWGGYVTYNDSVKGRFT

ISRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQ

GTLVTVSS

516
F010301090
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGWTTYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYQGQ

GTLVTVSS

517
F010301099
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGWVTYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

518
F010301111
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGYVNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYLGQ

GTLVTVSS

519
F010301113
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSVYQGQ

GTLVTVSS

520
F010301126
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYVTYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

521
F010301129
EVQLVESGGGLVQPGGSLRLSCAASGMLFNANTQ

GWYRQAPGKQRELVAFIFSGGWTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

522
F010301138
EVQLVESGGGLVQPGGSLRLSCAASGMLFYANTQ

GWYRQAPGKQRELVAFIFSGGYTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

523
F010301139
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFWFSGGYVNYNDSVKGRF

TISRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYQG

QGTLVTVSS

524
F010301162
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGWTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYQGQ

GTLVTVSS

525
F010301175
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYTTYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYLGQG

TLVTVSS

526
F010301188
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCSLSRYQGQ

GTLVTVSS

527
F010301191
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGWTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSVYQGQ

GTLVTVSS

528
F010301232
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYTTYVDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

529
F010301458
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGWTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

530
F010301463
EVQLVESGGGLVQPGGSLRLSCAASGMLFNRNTQ

GWYRQAPGKQRELVAFIFSGGYTNYNDSVKGRFTI

SRDNAKRTMYLQMNSLKPEDSAIYYCALSRYQGQ

GTLVTVSS

531
F010301301
EVQLVESGGGLVQAGGSLRLSCDASGRILRTGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

532
F010301304
EVQLVESGGGLVQAGGSLRLSCDASGRILRWGYM

RWHRQGAGKQREFVARITDDSATDYADSVKGRFTI

SRDNAKNTVYLQMNNLNPEDTAVYYCEALVTASV

RGGSIHSGTYWGRGTLVTVSS

533
F010301309
EVQLVESGGGLVQAGGSLRLSCDASGRIVRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

534
F010301313
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMK

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

535
F010301314
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

536
F010301328
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSAVDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

537
F010301335
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVAVITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

538
F010301344
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDGSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

539
F010301346
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

540
F010301350
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATVYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

541
F010301360
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSTTDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

542
F010301367
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITGDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

543
F010301372
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGGIHSGTYWGRGTLVTVSS

544
F010301387
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASAR

GGSIHSGTYWGRGTLVTVSS

545
F010301409
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIVSGTYWGRGTLVTVSS

546
F010301416
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGQYWGRGTLVTVSS

547
F010301418
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTWWGRGTLVTVSS

548
F010301425
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

VGSIHSGTYWGRGTLVTVSS

549
F010301440
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSTTYWGRGTLVTVSS

550
F010301445
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGYIHSGTYWGRGTLVTVSS

551
F010301557
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITGGSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

552
F010301558
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITGDSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

553
F010301559
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITDGSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

554
F010301560
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITGGSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

555
F010301561
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITGDSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

556
F010301562
EVQLVESGGGLVQAGGSLRLSCAASGRILRIGYMR

WHRQGAGKQREFVARITDGSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

557
F010301564
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITGGSATDYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

558
F010301565
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITGDSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

559
F010301566
EVQLVESGGGLVQAGGSLRLSCDASGRILRIGYMR

WHRQGAGKQREFVARITDGSATGYADSVKGRFTIS

RDNAKNTVYLQMNNLNPEDTAVYYCEALVTASVR

GGSIHSGTYWGRGTLVTVSS

560
F010301567
EVQLVESGGGLVQAGGSLRLSCAASVRPYSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

561
F010301568
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMT

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

562
F010301574
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMA

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

563
F010301578
EVQLVESGGGLVQAGGSLRLSCAASVRPFGTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

564
F010301579
EVQLVESGGGLVQAGGSLRLSCAASVKPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

565
F010301580
EVQLVESGGGLVQAGGSLRLSCAASVTPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

566
F010301584
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILTNGIVTYYADSVKGRFTIS

RDNAKNEVYLQMNKLKPEDTAVYYCALDRDYGG

RSFSAYEYEYWGQGTLVTVSS

567
F010301585
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIPTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

568
F010301586
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILANGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

569
F010301589
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGPVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

570
F010301591
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYK

GRSFSAYEYEYWGQGTLVTVSS

571
F010301592
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GSSFSAYEYEYWGQGTLVTVSS

572
F010301593
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDKDYG

GRSFSAYEYEYWGQGTLVTVSS

573
F010301594
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GKSFSAYEYEYWGQGTLVTVSS

574
F010301595
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRAYG

GRSFSAYEYEYWGQGTLVTVSS

575
F010301596
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYA

GRSFSAYEYEYWGQGTLVTVSS

576
F010301598
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYQ

GRSFSAYEYEYWGQGTLVTVSS

577
F010301604
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GQSFSAYEYEYWGQGTLVTVSS

578
F010301606
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAKEYEYWGQGTLVTVSS

579
F010301607
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAAEYEYWGQGTLVTVSS

580
F010301609
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAQEYEYWGQGTLVTVSS

581
F010301612
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSATEYEYWGQGTLVTVSS

582
F010301617
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSQSAYEYEYWGQGTLVTVSS

583
F010301618
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEHWGQGTLVTVSS

584
F010301619
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSKSAYEYEYWGQGTLVTVSS

585
F010301621
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAGEYEYWGQGTLVTVSS

586
F010301622
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAGILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSADEYEYWGQGTLVTVSS

587
F010301627
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVAAILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

588
F010301629
EVQLVESGGGLVQAGGSLRLSCAASVRPFSTSAMG

WFRQAPEKEREAVASILWNGIVTYYADSVKGRFTI

SRDNAKNEVYLQMNKLKPEDTAVYYCALDRDYG

GRSFSAYEYEYWGQGTLVTVSS

589
F010300316
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

WYRRAPGKQRELVASSTNGGSWNYADSVKGRFTI

SRDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYD

ISRTYWGQGTLVTVSS

590
F010300468
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

591
F010300477
EVQLVESGGGLVQPGGSLRLSCAASGSIFNINSMA

WYRRAPGKQRELVASSTNGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNWLLQPSIYDI

SRTYWGQGTLVTVSS

592
F010300631
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINSMA

WYRRAPGKQRELVAYSTPGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

593
F010300659
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINSMA

WYRRAPGKQRELVAYSTPGWDWNYADSVKGRFTI

SRDNAKRVYLQMNSLTPEDTAVYYCRWLLQPSIY

DISRTYWGQGTLVTVSS

594
F010300684
EVQLVESGGGLVQPGGSLRLSCAASGPVFNWNSM

AWYRRAPGKQRELVASSTPGGSTNYADSVKGRFTI

SRDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYD

ISRIYWGQGTLVTVSS

595
F010300796
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVAYSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRTYWGQGTLVTVSS

596
F010300880
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVASSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRWLLQPSIYDI

SRTYWGQGTLVTVSS

597
F010300900
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVASSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRWLLQPSIYDI

SRIYWGQGTLVTVSS

598
F010300948
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVASSTPGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRWLLQPSIYDI

SRIYWGQGTLVTVSS

599
F010300990
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVAYSTPGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRIYWGQGTLVTVSS

600
F010301000
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVAYSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCNALLQPSIYDI

SRIYWGQGTLVTVSS

601
F010301459
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVASSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

602
F010301460
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVAYSTPGGSTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

603
F010301462
EVQLVESGGGLVQPGGSLRLSCAASGPVFNINRMA

WYRRAPGKQRELVAYSTPGGDTNYADSVKGRFTIS

RDNAKRVYLQMNSLTPEDTAVYYCRALLQPSIYDI

SRTYWGQGTLVTVSS

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

	Number	Date	Country
	63271963	Oct 2021	US
	63115878	Nov 2020	US

NAV1.7 BINDERS

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