METHODS AND COMPOSITIONS RELATING TO GLP1R VARIANTS

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 22, 2021, is named 44854-808_201_SL.txt and is 838,237 bytes in size.

BACKGROUND

G protein-coupled receptors (GPCRs) are implicated in a wide variety of diseases. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigens because GPCRs are often expressed at low levels in cells and are very unstable when purified. Thus, there is a need for improved agents for therapeutic intervention which target GPCRs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies or antibody fragments that binds GLP1R, comprising an immunoglobulin heavy chain and an immunoglobulin light chain: (a) wherein the immunoglobulin heavy chain comprises an amino acid sequence at least about 90% identical to that set forth in Table 9; and (b) wherein the immunoglobulin light chain comprises an amino acid sequence at least about 90% identical to that set forth in Table 10. Further provided herein are antibodies or antibody fragments, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an agonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an antagonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an allosteric modulator of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the allosteric modulator of GLP1R is a negative allosteric modulator.

Provided herein are methods of treating a metabolic disease or disorder comprising administering an antibody or antibody fragment that binds GLP1R, wherein the antibody or antibody fragment comprises a sequence set forth in Tables 7-13. Further provided herein are methods, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are methods, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment is an agonist of GLP1R. Further provided herein are methods, wherein the antibody or antibody fragment is an antagonist of GLP1R. Further provided herein are methods, wherein the antibody or antibody fragment is an allosteric modulator of GLP1R. Further provided herein are methods, wherein the allosteric modulator of GLP1R is a negative allosteric modulator. Further provided herein are methods, wherein the antibody or antibody fragment is an allosteric modulator. Further provided herein are methods, wherein the antibody or antibody fragment is a negative allosteric modulator. Further provided herein are methods, wherein the metabolic disease or disorder is Type II diabetes or obesity.

Provided herein are antibodies or antibody fragments comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 441-619; (b) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 620-798; (c) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 799-977; (d) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 978-1156; (e) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 1157-1335; and (f) an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 1336-1514. Further provided herein are antibodies or antibody fragments, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an agonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an antagonist of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody fragment is an allosteric modulator of GLP1R. Further provided herein are antibodies or antibody fragments, wherein the allosteric modulator of GLP1R is a negative allosteric modulator. Further provided herein are antibodies or antibody fragments, wherein the VH comprises a sequence at least about 90% identical to any one of SEQ ID NOs: 58-77. Further provided herein are antibodies or antibody fragments, wherein the VH comprises a sequence of any one of SEQ ID NOs: 58-77. Further provided herein are antibodies or antibody fragments, wherein the VL comprises a sequence at least about 90% identical to any one of SEQ ID NOs: 92-111. Further provided herein are antibodies or antibody fragments, wherein the VL comprises a sequence of any one of SEQ ID NOs: 92-111.

Provided herein are methods of treating a metabolic disease or disorder comprising administering an antibody or antibody fragment that binds GLP1R comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 441-619; (b) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 620-798; (c) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 799-977; (d) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 978-1156; (e) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 1157-1335; and (f) an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 1336-1514. Further provided herein are methods, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are methods, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 20 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment has an EC50 less than about 10 nanomolar in a cAMP assay. Further provided herein are methods, wherein the antibody or antibody fragment is an agonist of GLP1R. Further provided herein are methods, wherein the antibody or antibody fragment is an antagonist of GLP1R. Further provided herein are methods, wherein the antibody or antibody fragment is an allosteric modulator of GLP1R. Further provided herein are methods, wherein the allosteric modulator of GLP1R is a negative allosteric modulator. Further provided herein are methods, wherein the antibody or antibody fragment is an allosteric modulator. Further provided herein are methods, wherein the antibody or antibody fragment is a negative allosteric modulator. Further provided herein are methods, wherein the VH comprises a sequence at least about 90% identical to any one of SEQ ID NOs: 58-77. Further provided herein are methods, wherein the VH comprises a sequence of any one of SEQ ID NOs: 58-77. Further provided herein are methods, wherein the VL comprises a sequence at least about 90% identical to any one of SEQ ID NOs: 92-111. Further provided herein are methods, wherein the VL comprises a sequence of any one of SEQ ID NOs: 92-111. Further provided herein are methods, wherein the metabolic disease or disorder is Type II diabetes or obesity.

Provided herein are nucleic acid compositions comprising: a) a first nucleic acid encoding a variable domain, heavy chain region (VH) comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and wherein (i) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 441-619; (ii) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 620-798; (iii) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 799-977; b) a second nucleic acid encoding a variable domain, light chain region (VL) comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (i) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 978-1156; (ii) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 1157-1335; and (iii) an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 1336-1514.

Provided herein are nucleic acid compositions comprising: a) a first nucleic acid encoding a variable domain, heavy chain region (VH) comprising an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 58-77; b) a second nucleic acid encoding a variable domain, light chain region (VL) comprising at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 92-111; and an excipient. Further provided herein are nucleic acid compositions, wherein the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 58-77. Further provided herein are nucleic acid compositions, wherein the VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 92-111. Further provided herein are nucleic acid compositions, wherein the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 58-77, and wherein the VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 92-111.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a first schematic of an immunoglobulin.

FIG. 1B depicts a second schematic of an immunoglobulin.

FIG. 2 depicts a schematic of a motif for placement in an immunoglobulin.

FIG. 3 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.

FIG. 4 illustrates an example of a computer system.

FIG. 5 is a block diagram illustrating an architecture of a computer system.

FIG. 6 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).

FIG. 7 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIG. 8A depicts a schematic of an immunoglobulin comprising a VH domain attached to a VL domain using a linker.

FIG. 8B depicts a schematic of a full-domain architecture of an immunoglobulin comprising a VH domain attached to a VL domain using a linker, a leader sequence, and pIII sequence.

FIG. 8C depicts a schematic of four framework elements (FW1, FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

FIG. 9A depicts a structure of Glucagon-like peptide 1 (GLP-1, cyan) in complex with GLP-1 receptor (GLP-1R, grey), PDB entry 5VAI.

FIG. 9B depicts a crystal structure of CXCR4 chemokine receptor (grey) in complex with a cyclic peptide antagonist CVX15 (blue), PDB entry 30R0.

FIG. 9C depicts a crystal structure of human smoothened receptor with the transmembrane domain in grey and extracellular domain (ECD) in orange, PDB entry 5L7D. The ECD contacts the TMD through extracellular loop 3 (ECL3).

FIG. 9D depicts a structure of GLP-1R (grey) in complex with a Fab (magenta), PDB entry 6LN2.

FIG. 9E depicts a crystal structure of CXCR4 (grey) in complex with a viral chemokine antagonist Viral macrophage inflammatory protein 2 (vMIP-II, green), PDB entry 4RWS.

FIG. 10 depicts a schema of the GPCR focused library design. Two germline heavy chain VH1-69 and VH3-30; 4 germline light chain IGKV1-39 and IGKV3-15, and IGLV1-51 and IGLV2-14.

FIG. 11 depicts a graph of HCDR3 length distribution in the GPCR-focused library compared to the HCDR3 length distribution in B-cell populations from three healthy adult donors. In total, 2,444,718 unique VH sequences from the GPCR library and 2,481,511 unique VH sequences from human B-cell repertoire were analyzed to generate the length distribution plot.

FIG. 12A depicts the design of the over-expressing GLP-1R CHO cells for the phage antibody library selection. GLP-1R expression was confirmed by the gating of double detection of GFP green fluorescence and the surface expression of Flag tag on the cell surface.

FIG. 12B depicts a cell-based panning process.

FIG. 13 depicts a graph of percent unique HCDR3 in the output pools of the five GLP-1R panning rounds.

FIG. 14 depicts a graph of binding plots of the 13 unique GLP-1R Hits, compared to the parental CHO cell binding.

FIG. 15 depicts HCDR3 loop sequences of the 13 unique GLP1R binders. Six of the clones have a GLP-1 motif, four of the clones have a GLP-2 motif, and three clones do not have a GLP-1 or GLP-2 motif. For the clones that have the GLP-1 or GLP-2 motif, residues that are similar to the GLP-1 sequence or the GLP-2 sequence are colored in black and the residues that are different are colored red. Functional antagonists in the cAMP assay are highlighted in yellow. FIG. 15 discloses SEQ ID NOS 1528, 1-2, 27, 12, 3, 32, 1529, 23, 25, 30, 1530, 19, 22 and 24, respectively, in order of appearance.

FIG. 16A depicts a graph of orthosteric inhibition of GLP1R-3 binding in the absence and presence of GLP-1 (7-36).

FIG. 16B depicts a graph of effects of GLP1R-3 on GLP-1 activation in the cAMP assay.

FIG. 16C depicts a graph of effects of GLP1R-3 on GLP-1 induced @-arrestin recruitment.

FIG. 17 depicts a design of GLP1R-59-2. The GLP1 (7-36) peptide was linked to the N-terminal of light chain of the functionally inactive GLP-1R binding antibody GLP1R-2.

FIG. 18A depicts a graph of GLP1R-59-2 binding specifically to the GLP-1R with an EC50 of 15.5 nM.

FIG. 18B depicts a graph of GLP1R-59-2 in the cAMP assay with a similar EC50 as the GLP-1 7-36 peptide.

FIG. 18C depicts a graph of GLP1R-59-2 on inducing the β-arrestin recruitment in GLP-1R expression cells.

FIGS. 19A-19B depict in vivo pharmacokinetic (PK) and pharmacodynamic (PD) effects of GLP1R-3 and GLP1R-59-2. Based on the beta phase calculation, GLP1R-3 has a 1-week half-life in rat (FIG. 19A). GLP1R-59-2 has a 2-day half-life in rat (FIG. 19B).

FIG. 20A depicts a graph of GLP1R-59-2 on glucose after glucose challenge.

FIG. 20B depicts a graph of Area Under the Curve (AUC) in a glucose tolerance test (GTT).

FIG. 21A depicts a graph of GLP1R-3 and GLP-1 peptide Exendin 9-39 treatment, 19+2 hour dosing regimen

FIG. 21B depicts a graph of Area Under the Curve (AUC) in an insulin tolerance test (ITT).

FIG. 22A depicts a graph of GLP1R-3 treatment, single 6 hour dosing regimen after insulin challenge, as compared to GLP-1 peptide Exendin 9-39 (1.0 or 0.23 mg/kg dose) or control.

FIG. 22B depicts a graph of Area Under the Curve (AUC) of GLP1R-3 (20 mg/kg) treatment at 6 hours in an ITT.

FIG. 23A depicts a graph of GLP1R-3 treatment, single 6 hour dosing regimen after insulin challenge, as compared to GLP1R-226-1, GLP1R-226-2, or control.

FIG. 23B depicts a graph Area Under the Curve (AUC) of GLP1R-3 treatment, single 6 hour dosing regimen after insulin challenge, as compared to GLP1R-226-1, GLP1R-226-2, or control.

FIGS. 24A-24B are schemas of panning strategy for GLP1R-221 and GLP1R-222 variants.

FIGS. 25A-25B are graphs of competition data for GLP1R-221 and GLP1R-222 variants.

FIG. 26 is a graph of GLP1R-221 and GLP1R-222 variants in a cAMP assay.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

Definitions

Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, components, and/or groups, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.

GPCR Libraries for GLP1 Receptor

Provided herein are methods and compositions relating to G protein-coupled receptor (GPCR) binding libraries for glucagon-like peptide-1 receptor (GLP1R) comprising nucleic acids encoding for an immunoglobulin comprising a GPCR binding domain. Immunoglobulins as described herein can stably support a GPCR binding domain. The GPCR binding domain may be designed based on surface interactions of a GLP1R ligand and GLP1R. Libraries as described herein may be further variegated to provide for variant libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries that may be generated when the nucleic acid libraries are translated. In some instances, nucleic acid libraries as described herein are transferred into cells to generate a cell library. Also provided herein are downstream applications for the libraries synthesized using methods described herein. Downstream applications include identification of variant nucleic acids or protein sequences with enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and for the treatment or prevention of a disease state associated with GPCR signaling.

Provided herein are libraries comprising nucleic acids encoding for an immunoglobulin. In some instances, the immunoglobulin is an antibody. As used herein, the term antibody will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CH1 domains), a F(ab′)2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CH1 fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an immunoglobulin, wherein the immunoglobulin is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an immunoglobulin, wherein the immunoglobulin is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2), or subclass.

In some embodiments, libraries comprise immunoglobulins that are adapted to the species of an intended therapeutic target. Generally, these methods include “mammalization” and comprise methods for transferring donor antigen-binding information to a less immunogenic mammal antibody acceptor to generate useful therapeutic treatments. In some instances, the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, or human. In some instances, provided herein are libraries and methods for felinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. In some instances, these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canine antigen-binding information from a donor antibody to a less immunogenic canine antibody acceptor to generate treatments useful as therapeutics in dogs. In some instances, caninized forms of non-canine antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-canine antibodies. In some instances, caninized antibodies are canine antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-canine species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the canine antibody are replaced by corresponding non-canine FR residues. In some instances, caninized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The caninized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a canine antibody.

“Felinization” can comprise a method for transferring non-feline antigen-binding information from a donor antibody to a less immunogenic feline antibody acceptor to generate treatments useful as therapeutics in cats. In some instances, felinized forms of non-feline antibodies provided herein are chimeric antibodies that contain minimal sequence derived from non-feline antibodies. In some instances, felinized antibodies are feline antibody sequences (“acceptor” or “recipient” antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-feline species (“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks, non-human primates, human, humanized, recombinant sequence, or an engineered sequence having the desired properties. In some instances, framework region (FR) residues of the feline antibody are replaced by corresponding non-feline FR residues. In some instances, felinized antibodies include residues that are not found in the recipient antibody or in the donor antibody. In some instances, these modifications are made to further refine antibody performance. The felinized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc) of a felinize antibody.

Provided herein are libraries comprising nucleic acids encoding for a non-immunoglobulin. For example, the non-immunoglobulin is an antibody mimetic. Exemplary antibody mimetics include, but are not limited to, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins, fynomers, Kunitz domain-based proteins, monobodies, anticalins, knottins, armadillo repeat protein-based proteins, and bicyclic peptides.

Libraries described herein comprising nucleic acids encoding for an immunoglobulin comprising variations in at least one region of the immunoglobulin. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domain including, but not limited to, CDRL1, CDRL2, and CDRL3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for an immunoglobulin, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the variant library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin for variation is from heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain V-gene family, or light chain J-gene family. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary genes include, but are not limited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In some instances, the gene is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the gene is IGHJ3, IGHJ6, IGHJ, or IGHJ4.

Provided herein are libraries comprising nucleic acids encoding for immunoglobulins, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the immunoglobulin libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for immunoglobulins as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the immunoglobulins comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

A number of variant sequences for the at least one region of the immunoglobulin for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000 sequences. In some instances, the number of variant sequences is about 10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.

Variant sequences for the at least one region of the immunoglobulin, in some instances, vary in length or sequence. In some instances, the at least one region that is de novo synthesized is for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances, the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about 10¹, 10², 103, 104, 105, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰variants.

Following synthesis of libraries described herein, libraries may be used for screening and analysis. For example, libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof. In some instances, the libraries are assayed for immunoglobulin (e.g., an antibody) capable of folding. In some instances, a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. For example, a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.

GLP1R Libraries

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for immunoglobulins (e.g., antibodies) that bind to GLP1R. In some instances, the immunoglobulin sequences for GLP1R binding domains are determined by interactions between the GLP1R binding domains and the GLP1R.

Provided herein are libraries comprising nucleic acids encoding immunoglobulins comprising GLP1R binding domains, wherein the GLP1R binding domains are designed based on surface interactions on GLP1R. In some instances, the GLP1R comprises a sequence as defined by SEQ ID NO: 1. In some instances, the GLP1R binding domains interact with the amino- or carboxy-terminus of the GLP1R. In some instances, the GLP1R binding domains interact with at least one transmembrane domain including, but not limited to, transmembrane domain 1 (TM1), transmembrane domain 2 (TM2), transmembrane domain 3 (TM3), transmembrane domain 4 (TM4), transmembrane domain 5 (TM5), transmembrane domain 6 (TM6), and transmembrane domain 7 (TM7). In some instances, the GLP1R binding domains interact with an intracellular surface of the GLP1R. For example, the GLP1R binding domains interact with at least one intracellular loop including, but not limited to, intracellular loop 1 (ICL1), intracellular loop 2 (ICL2), and intracellular loop 3 (ICL3). In some instances, the GLP1R binding domains interact with an extracellular surface of the GLP1R. For example, the GLP1R binding domains interact with at least one extracellular domain (ECD) or extracellular loop (ECL) of the GLP1R. The extracellular loops include, but are not limited to, extracellular loop 1 (ECL1), extracellular loop 2 (ECL2), and extracellular loop 3 (ECL3).

Described herein are GLP1R binding domains, wherein the GLP1R binding domains are designed based on surface interactions between a GLP1R ligand and the GLP1R. In some instances, the ligand is a peptide. In some instances, the ligand is glucagon, glucagon-like peptide 1-(7-36)amide, glucagon-like peptide 1-(7-37), liraglutide, exendin-4, lixisenatide, T-0632, GLP1R0017, or BETP. In some instances, the ligand is a GLP1R agonist. In some instances, the ligand is a GLP1R antagonist. In some instances, the ligand is a GLP1R allosteric modulator. In some instances, the allosteric modulator is a negative allosteric modulator. In some instances, the allosteric modulator is a positive allosteric modulator.

Sequences of GLP1R binding domains based on surface interactions between a GLP1R ligand and the GLP1R are analyzed using various methods. For example, multispecies computational analysis is performed. In some instances, a structure analysis is performed. In some instances, a sequence analysis is performed. Sequence analysis can be performed using a database known in the art. Non-limiting examples of databases include, but are not limited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC Genome Browser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPS Guide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are GLP1R binding domains designed based on sequence analysis among various organisms. For example, sequence analysis is performed to identify homologous sequences in different organisms. Exemplary organisms include, but are not limited to, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human.

Following identification of GLP1R binding domains, libraries comprising nucleic acids encoding for the GLP1R binding domains may be generated. In some instances, libraries of GLP1R binding domains comprise sequences of GLP1R binding domains designed based on conformational ligand interactions, peptide ligand interactions, small molecule ligand interactions, extracellular domains of GLP1R, or antibodies that target GLP1R. In some instances, libraries of GLP1R binding domains comprise sequences of GLP1R binding domains designed based on peptide ligand interactions. Libraries of GLP1R binding domains may be translated to generate protein libraries. In some instances, libraries of GLP1R binding domains are translated to generate peptide libraries, immunoglobulin libraries, derivatives thereof, or combinations thereof. In some instances, libraries of GLP1R binding domains are translated to generate protein libraries that are further modified to generate peptidomimetic libraries. In some instances, libraries of GLP1R binding domains are translated to generate protein libraries that are used to generate small molecules.

Methods described herein provide for synthesis of libraries of GLP1R binding domains comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the libraries of GLP1R binding domains comprise varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GLP1R binding domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GLP1R binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for the GLP1R binding domains, wherein the libraries comprise sequences encoding for variation of length of the GLP1R binding domains. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Following identification of GLP1R binding domains, the GLP1R binding domains may be placed in immunoglobulins as described herein. In some instances, the GLP1R binding domains are placed in the CDRH3 region. GPCR binding domains that may be placed in immunoglobulins can also be referred to as a motif Immunoglobulins comprising GLP1R binding domains may be designed based on binding, specificity, stability, expression, folding, or downstream activity. In some instances, the immunoglobulins comprising GLP1R binding domains enable contact with the GLP1R. In some instances, the immunoglobulins comprising GLP1R binding domains enables high affinity binding with the GLP1R. An exemplary amino acid sequence of GLP1R binding domain is described in Table 1.

TABLE 1

GLP1R amino acid sequences

SEQ

ID NO
GPCR
Amino Acid Sequence

1352
GLP1R
RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYA

CWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNS

SLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIASAIL

LGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQ

WDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSV

LSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNY

WLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLT

LIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCF

VNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGS

SMYTATCQASCS

Provided herein are immunoglobulins comprising GLP1R binding domains, wherein the sequences of the GLP1R binding domains support interaction with GLP1R. The sequence may be homologous or identical to a sequence of a GLP1R ligand. In some instances, the GLP1R binding domain sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 95% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 97% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 99% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least or about 100% homology to SEQ ID NO: 1. In some instances, the GLP1R binding domain sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NO: 1.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term “homology” or “similarity” between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein sequence to the second protein sequence. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).

The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDRH1, CDRH2, CDRH3) and three CDRs in each light chain variable region (CDRL1, CDRL2, CDRL3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Whitelegg N R and Rees A R, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 December; 13(12):819-24 (“AbM” numbering scheme. In certain embodiments the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for immunoglobulins comprising GLP1R binding domains comprise variation in domain type, domain length, or residue variation. In some instances, the domain is a region in the immunoglobulin comprising the GLP1R binding domains. For example, the region is the VH, CDRH3, or VL domain. In some instances, the domain is the GLP1R binding domain.

Methods described herein provide for synthesis of a GLP1R binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the GLP1R binding library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a VH, CDRH3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GLP1R binding domain. For example, at least one single codon of a GLP1R binding domain as listed in Table 1 is varied. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a VH, CDRH3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GLP1R binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of a GLP1R binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence, wherein the GLP1R binding library comprises sequences encoding for variation of length of a domain. In some instances, the domain is VH, CDRH3, or VL domain. In some instances, the domain is the GLP1R binding domain. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for immunoglobulins comprising GLP1R binding domains, wherein the GLP1R binding libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the VH, CDRH3, or VL domain. In some instances, the GLP1R binding libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

GLP1R binding libraries comprising nucleic acids encoding for immunoglobulins comprising GLP1R binding domains as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 to about 75 amino acids.

GLP1R binding libraries comprising de novo synthesized variant sequences encoding for immunoglobulins comprising GLP1R binding domains comprise a number of variant sequences. In some instances, a number of variant sequences is de novo synthesized for a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or a combination thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, a number of variant sequences is de novo synthesized for a GPCR binding domain. For example, the number of variant sequences is about 1 to about 10 sequences for the VH domain, about 108 sequences for the GLP1R binding domain, and about 1 to about 44 sequences for the VK domain. The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is about 10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150 sequences.

Described herein are antibodies or antibody fragments thereof that binds GLP1R. In some embodiments, the antibody or antibody fragment thereof comprises a sequence as set forth in Tables 7-13. In some embodiments, the antibody or antibody fragment thereof comprises a sequence that is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forth in Tables 7-13.

In some instances, an antibody or antibody fragment described herein comprises a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, an antibody or antibody fragment described herein comprises a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, an antibody or antibody fragment described herein comprises a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977.

In some instances, an antibody or antibody fragment described herein comprises a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, an antibody or antibody fragment described herein comprises a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, an antibody or antibody fragment described herein comprises a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 80% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 85% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 90% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, an antibody or antibody fragment described herein comprises a sequence that is at least 95% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514.

In some embodiments, the antibody or antibody fragment comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 441-619; (b) an amino acid sequence of CDRH2 is as set forth in any one of SEQ ID NOs: 620-798; (c) an amino acid sequence of CDRH3 is as set forth in any one of SEQ ID NOs: 799-977; (d) an amino acid sequence of CDRL1 is as set forth in any one of SEQ ID NOs: 978-1156; (e) an amino acid sequence of CDRL2 is as set forth in any one of SEQ ID NOs: 1157-1335; and (f) an amino acid sequence of CDRL3 is as set forth in any one of SEQ ID NOs: 1336-1514. In some embodiments, the antibody or antibody fragment comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein VH comprises complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino acid sequence of CDRH1 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 441-619; (b) an amino acid sequence of CDRH2 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 620-798; (c) an amino acid sequence of CDRH3 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 799-977; (d) an amino acid sequence of CDRL1 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 978-1156; (e) an amino acid sequence of CDRL2 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1157-1335; and (f) an amino acid sequence of CDRL3 is at least or about 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1336-1514.

Described herein, in some embodiments, are antibodies or antibody fragments comprising a variable domain, heavy chain region (VH) and a variable domain, light chain region (VL), wherein the VH comprises an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 58-77, and wherein the VL comprises an amino acid sequence at least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 92-111. In some instances, the antibodies or antibody fragments comprise VH comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 58-77, and VL comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 92-111.

The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Typically, techniques for determining sequence identity include comparing two nucleotide or amino acid sequences and the determining their percent identity. Sequence comparisons, such as for the purpose of assessing identities, may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters. The “percent identity”, also referred to as “percent homology”, between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). High sequence identity generally includes ranges of sequence identity of approximately 80% to 100% and integer values there between.

GLP1R binding libraries comprising de novo synthesized variant sequences encoding for immunoglobulins comprising GLP1R binding domains comprise improved diversity. For example, variants are generated by placing GLP1R binding domain variants in immunoglobulins comprising N-terminal CDRH3 variations and C-terminal CDRH3 variations. In some instances, variants include affinity maturation variants. Alternatively or in combination, variants include variants in other regions of the immunoglobulin including, but not limited to, CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3. In some instances, the number of variants of the GLP1R binding libraries is at least or about 10⁴, 10⁵, 10⁶, 10⁷, 108, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰non-identical sequences. For example, a library comprising about 10 variant sequences for a VH region, about 237 variant sequences for a CDRH3 region, and about 43 variant sequences for a VL and CDRL3 region comprises 10⁵non-identical sequences (10×237×43).

In some instances, the at least one region of the antibody for variation is from heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain V-gene family, or light chain J-gene family. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary regions of the antibody for variation include, but are not limited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1. In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the gene is IGHV1-69 and IGHV3-30. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the region of the antibody for variation is IGHJ3, IGHJ6, IGHJ, or IGHJ4. In some instances, the at least one region of the antibody for variation is IGHV1-69, IGHV3-23, IGKV3-20, IGKV1-39, or combinations thereof. In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV3-20, In some instances, the at least one region of the antibody for variation is IGHV1-69 and IGKV1-39. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV3-20. In some instances, the at least one region of the antibody for variation is IGHV3-23 and IGKV1-39.

Provided herein are libraries comprising nucleic acids encoding for a GLP1R antibody comprising variation in at least one region of the antibody, wherein the region is the CDR region. In some instances, the GLP1R antibody is a single domain antibody comprising one heavy chain variable domain such as a VHH antibody. In some instances, the VHH antibody comprises variation in one or more CDR regions. In some instances, libraries described herein comprise at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3. In some instances, libraries described herein comprise at least or about 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, or more than 10²⁰sequences of a CDR1, CDR2, or CDR3. For example, the libraries comprise at least 2000 sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600 sequences for CDR3. In some instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain (VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. In some instances, libraries described herein comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3 of the VL. In some instances, libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences of a CDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise at least 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2 of the VL, and at least 140 sequences of a CDR3 of the VL. In some instances, the libraries comprise at least 2 sequences of a CDR1 of the VL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequences of a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In some instances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain (VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In some instances, libraries described herein comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3 of the VH. In some instances, libraries described herein comprise at least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 1015, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences of a CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise at least 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2 of the VH, and at least 10⁸sequences of a CDR3 of the VH. In some instances, the libraries comprise at least 30 sequences of a CDR1 of the VH, at least 860 sequences of a CDR2 of the VH, and at least 10⁷sequences of a CDR3 of the VH. In some instances, the VH is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. In some instances, the VH is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the VH is IGHV1-69 or IGHV3-30. In some instances, the VH is IGHV3-23.

Libraries as described herein, in some embodiments, comprise varying lengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3. In some instances, the length of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acids in length. For example, the CDRH3 comprises at least or about 12, 15, 16, 17, 20, 21, or 23 amino acids in length. In some instances, the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprises a range of about 1 to about 10, about 5 to about 15, about 10 to about 20, or about 15 to about 30 amino acids in length.

Libraries comprising nucleic acids encoding for antibodies having variant CDR sequences as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the antibodies comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

Ratios of the lengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 may vary in libraries described herein. In some instances, a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprising at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acids in length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the library. For example, a CDRH3 comprising about 23 amino acids in length is present in the library at 40%, a CDRH3 comprising about 21 amino acids in length is present in the library at 30%, a CDRH3 comprising about 17 amino acids in length is present in the library at 20%, and a CDRH3 comprising about 12 amino acids in length is present in the library at 10%. In some instances, a CDRH3 comprising about 20 amino acids in length is present in the library at 40%, a CDRH3 comprising about 16 amino acids in length is present in the library at 30%, a CDRH3 comprising about 15 amino acids in length is present in the library at 20%, and a CDRH3 comprising about 12 amino acids in length is present in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprise variant CDR sequences that are shuffled to generate a library with a theoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 1020 sequences. In some instances, the library has a final library diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 1020 sequences.

Provided herein are GLP1R binding libraries encoding for an immunoglobulin. In some instances, the GLP1R immunoglobulin is an antibody. In some instances, the GLP1R immunoglobulin is a VHH antibody. In some instances, the GLP1R immunoglobulin comprises a binding affinity (e.g., kD) to GLP1R of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 1 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 1.2 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 2 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 5 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 10 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 13.5 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 15 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 20 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 25 nM. In some instances, the GLP1R immunoglobulin comprises a kD of less than 30 nM.

In some instances, the GLP1R immunoglobulin is a GLP1R agonist. In some instances, the GLP1R immunoglobulin is a GLP1R antagonist. In some instances, the GLP1R immunoglobulin is a GLP1R allosteric modulator. In some instances, the allosteric modulator is a negative allosteric modulator. In some instances, the allosteric modulator is a positive allosteric modulator. In some instances, the GLP1R immunoglobulin results in agonistic, antagonistic, or allosteric effects at a concentration of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, or more than 1000 nM. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator at a concentration of at least or about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. In some instances, the GLP1R immunoglobulin is a negative allosteric modulator at a concentration in a range of about 0.001 to about 100, 0.01 to about 90, about 0.1 to about 80, 1 to about 50, about 10 to about 40 nM, or about 1 to about 10 nM. In some instances, the GLP1R immunoglobulin comprises an EC50 or IC50 of at least or about 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.06, 0.07, 0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6 nM. In some instances, the GLP1R immunoglobulin comprises an EC50 or IC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM.

Provided herein are GLP1R binding libraries encoding for an immunoglobulin, wherein the immunoglobulin comprises a long half-life. In some instances, the half-life of the GLP1R immunoglobulin is at least or about 12 hours, 24 hours 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some instances, the half-life of the GLP1R immunoglobulin is in a range of about 12 hours to about 300 hours, about 20 hours to about 280 hours, about 40 hours to about 240 hours, or about 60 hours to about 200 hours.

GLP1R immunoglobulins as described herein may comprise improved properties. In some instances, the GLP1R immunoglobulins are monomeric. In some instances, the GLP1R immunoglobulins are not prone to aggregation. In some instances, at least or about 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the GLP1R immunoglobulins are monomeric. In some instances, the GLP1R immunoglobulins are thermostable. In some instances, the GLP1R immunoglobulins result in reduced non-specific binding.

Following synthesis of GLP1R binding libraries comprising nucleic acids encoding immunoglobulins comprising GLP1R binding domains, libraries may be used for screening and analysis. For example, libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, the GLP1R binding libraries comprises nucleic acids encoding immunoglobulins comprising GPCR binding domains with multiple tags such as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances, libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding for immunoglobulins comprising GLP1R binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity. In some instances, libraries described herein are used for screening and analysis.

Provided herein are libraries comprising nucleic acids encoding for immunoglobulins comprising GLP1R binding domains, wherein the nucleic acid libraries are used for screening and analysis. In some instances, screening and analysis comprise in vitro, in vivo, or ex vivo assays. Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect. In some instances, cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In some instances, nucleic acid libraries described herein may also be delivered to a multicellular organism. Exemplary multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries or protein libraries encoded thereof described herein may be screened for various pharmacological or pharmacokinetic properties. In some instances, the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays. For example, in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity. Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity properties, immunogenicity, potency, and clinical safety properties.

Pharmacological or pharmacokinetic properties that may be screened include, but are not limited to, cell binding affinity and cell activity. For example, cell binding affinity assays or cell activity assays are performed to determine agonistic, antagonistic, or allosteric effects of libraries described herein. In some instances, the cell activity assay is a cAMP assay. In some instances, libraries as described herein are compared to cell binding or cell activity of ligands of GLP1R.

Libraries as described herein may be screened in cell-based assays or in non-cell-based assays. Examples of non-cell-based assays include, but are not limited to, using viral particles, using in vitro translation proteins, and using protealiposomes with GLP1R.

Nucleic acid libraries as described herein may be screened by sequencing. In some instances, next generation sequence is used to determine sequence enrichment of GLP1R binding variants. In some instances, V gene distribution, J gene distribution, V gene family, CDR3 counts per length, or a combination thereof is determined. In some instances, clonal frequency, clonal accumulation, lineage accumulation, or a combination thereof is determined. In some instances, number of sequences, sequences with VH clones, clones, clones greater than 1, clonotypes, clonotypes greater than 1, lineages, simpsons, or a combination thereof is determined. In some instances, a percentage of non-identical CDR3s is determined. For example, the percentage of non-identical CDR3s is calculated as the number of non-identical CDR3s in a sample divided by the total number of sequences that had a CDR3 in the sample.

Provided herein are nucleic acid libraries, wherein the nucleic acid libraries may be expressed in a vector. Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors. Exemplary expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3×FLAG, pSF-CMV-NEO-COOH-3×FLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-N1 Vector, pEF1a-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A and pDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in a vector to generate a construct comprising an immunoglobulin comprising sequences of GLP1R binding domains. In some instances, a size of the construct varies. In some instances, the construct comprises at least or about 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some instances, a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding for immunoglobulins comprising GPCR binding domains, wherein the nucleic acid libraries are expressed in a cell. In some instances, the libraries are synthesized to express a reporter gene. Exemplary reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein, cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.

Diseases and Disorders

Provided herein are GLP1R binding libraries comprising nucleic acids encoding for immunoglobulins (e.g., antibodies) comprising GLP1R binding domains that may have therapeutic effects. In some instances, the GLP1R binding libraries result in protein when translated that is used to treat a disease or disorder. In some instances, the protein is an immunoglobulin. In some instances, the protein is a peptidomimetic.

GLP1R libraries as described herein may comprise modulators of GLP1R. In some instances, the modulator of GLP1R is an inhibitor. In some instances, the modulator of GLP1R is an activator. In some instances, the GLP1R inhibitor is a GLP1R antagonist. In some instances, the GLP1R antagonist is GLP1R-3. Modulators of GLP1R, in some instances, are used for treating various diseases or disorders.

Exemplary diseases include, but are not limited to, cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, or a neurological disease or disorder. In some instances, the cancer is a solid cancer or a hematologic cancer. In some instances, a modulator of GLP1R as described herein is used for treatment of weight gain (or for inducing weight loss), treatment of obesity, or treatment of Type II diabetes. In some instances, the GLP1R modulator is used for treating hypoglycemia. In some instances, the GLP1R modulator is used for treating post-bariatric hypoglycemia. In some instances, the GLP1R modulator is used for treating severe hypoglycemia. In some instances, the GLP1R modulator is used for treating hyperinsulinism. In some instances, the GLP1R modulator is used for treating congenital hyperinsulinism.

In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously.

Described herein are pharmaceutical compositions comprising antibodies or antibody fragment thereof that binds GLP1R. In some embodiments, the antibody or antibody fragment thereof comprises a sequence as set forth in Tables 7-13. In some embodiments, the antibody or antibody fragment thereof comprises a sequence that is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence as set forth in Tables 7-13.

In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRH1 sequence of any one of SEQ ID NOs: 441-619. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRH2 sequence of any one of SEQ ID NOs: 620-798. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRH3 sequence of any one of SEQ ID NOs: 799-977.

In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRL1 sequence of any one of SEQ ID NOs: 978-1156. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRL2 sequence of any one of SEQ ID NOs: 1157-1168. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRL2 sequence of any one of SEQ ID NOs: 1157-1335. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 80% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 85% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 90% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a sequence that is at least 95% identical to a CDRL3 sequence of any one of SEQ ID NOs: 1336-1514.

Described herein are pharmaceutical compositions comprising antibodies or antibody fragment thereof that binds GLP1R that comprise various dosages of the antibodies or antibody fragment. In some instances, the dosage is ranging from about 1 to 80 mg/kg, from about 1 to about 100 mg/kg, from about 5 to about 100 mg/kg, from about 5 to about 80 mg/kg, from about 5 to about 60 mg/kg, from about 5 to about 50 mg/kg or from about 5 to about 500 mg/kg which can be administered in single or multiple doses. In some instances, the dosage is administered in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.10 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105 mg/kg, about 110 mg/kg, about 115 mg/kg, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 240, about 250, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360 mg/kg, about 370 mg/kg, about 380 mg/kg, about 390 mg/kg, about 400 mg/kg, 410 mg/kg, about 420 mg/kg, about 430 mg/kg, about 440 mg/kg, about 450 mg/kg, about 460 mg/kg, about 470 mg/kg, about 480 mg/kg, about 490 mg/kg, or about 500 mg/kg.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence. In some instances, each nucleic acid of a first nucleic acid population contains a variant at a single variant site. In some instances, the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position. The first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position. Each variant may encode for a codon that results in a different amino acid during translation. Table 2 provides a listing of each codon possible (and the representative amino acid) for a variant site.

TABLE 2

List of codons and amino acids

One
Three

letter
letter

Amino Acids
code
code
Codons

Alanine
A
Ala
GCA
GCC
GCG
GCT

Cysteine
C
Cys
TGC
TGT

Aspartic acid
D
Asp
GAC
GAT

Glutamic acid
E
Glu
GAA
GAG

Phenylalanine
F
Phe
TTC
TTT

Glycine
G
Gly
GGA
GGC
GGG
GGT

Histidine
H
His
CAC
CAT

Isoleucine
I
Iso
ATA
ATC
ATT

Lysine
K
Lys
AAA
AAG

Leucine
L
Leu
TTA
TTG
CTA
CTC
CTG
CTT

Methionine
M
Met
ATG

Asparagine
N
Asn
AAC
AAT

Proline
P
Pro
CCA
CCC
CCG
CCT

Glutamine
Q
Gln
CAA
CAG

Arginine
R
Arg
AGA
AGG
CGA
CGC
CGG
CGT

Serine
S
Ser
AGC
AGT
TCA
TCC
TCG
TCT

Threonine
T
Thr
ACA
ACC
ACG
ACT

Valine
V
Val
GTA
GTC
GTG
GTT

Tryptophan
W
Trp
TGG

Tyrosine
Y
Tyr
TAC
TAT

A nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a single nucleic acid. In some instances, each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single long nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform. Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomic data has become an important factor for studies that delve into the biological roles of various genes in both normal biology and disease pathogenesis. At the core of this research is the central dogma of molecular biology and the concept of “residue-by-residue transfer of sequential information.” Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.

Another exciting area of study is on the discovery, development and manufacturing of therapeutic molecules focused on a highly-specific cellular target. High diversity DNA sequence libraries are at the core of development pipelines for targeted therapeutics. Gene mutants are used to express proteins in a design, build, and test protein engineering cycle that ideally culminates in an optimized gene for high expression of a protein with high affinity for its therapeutic target. As an example, consider the binding pocket of a receptor. The ability to test all sequence permutations of all residues within the binding pocket simultaneously will allow for a thorough exploration, increasing chances of success. Saturation mutagenesis, in which a researcher attempts to generate all possible mutations at a specific site within the receptor, represents one approach to this development challenge. Though costly and time- and labor-intensive, it enables each variant to be introduced into each position. In contrast, combinatorial mutagenesis, where a few selected positions or short stretch of DNA may be modified extensively, generates an incomplete repertoire of variants with biased representation.

To accelerate the drug development pipeline, a library with the desired variants available at the intended frequency in the right position available for testing—in other words, a precision library—enables reduced costs as well as turnaround time for screening. Provided herein are methods for synthesizing nucleic acid synthetic variant libraries which provide for precise introduction of each intended variant at the desired frequency. To the end user, this translates to the ability to not only thoroughly sample sequence space but also be able to query these hypotheses in an efficient manner, reducing cost and screening time. Genome-wide editing can elucidate important pathways, libraries where each variant and sequence permutation can be tested for optimal functionality, and thousands of genes can be used to reconstruct entire pathways and genomes to re-engineer biological systems for drug discovery.

In a first example, a drug itself can be optimized using methods described herein. For example, to improve a specified function of an antibody, a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized. A variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector). The antibody is then expressed in a production cell line and screened for enhanced activity. Example screens include examining modulation in binding affinity to an antigen, stability, or effector function (e.g., ADCC, complement, or apoptosis). Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (VH or VL), and specific complementarity-determining regions (CDRs) of VH or VL.

Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state. Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system. Exemplary model systems include, without limitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction or treatment of a disease state, a variant nucleic acid library described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced. In some instances, an agent is used to induce a disease state in cells. Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia. The cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition. Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer). In some instances, the variant nucleic acid library is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity. Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity, and aging, response to free radical damage, or any combination thereof.

Substrates

Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides. In some instances, substrates comprise a homogenous array surface. For example, the homogenous array surface is a homogenous plate. The term “locus” as used herein refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface. In some instances, a locus is on a two-dimensional surface, e.g., a substantially planar surface. In some instances, a locus is on a three-dimensional surface, e.g., a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.

Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some instances, a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the surfaces provide support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the growth of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinct loci of a substrate, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some instances, a substrate comprises about 10,000 distinct loci. The amount of loci within a single cluster is varied in different instances. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances, each cluster includes about 50-500 loci. In some instances, each cluster includes about 100-200 loci. In some instances, each cluster includes about 100-150 loci. In some instances, each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In some cases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um. In some instances, the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4 clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50 clusters per 1 mm²or more. In some instances, a substrate comprises from about 1 cluster per 10 mm²to about 10 clusters per 1 mm². In some instances, the distance between the centers of two adjacent clusters is at least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm. In some instances, the diameter of a substrate is between about 25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In some instances, a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm²or more. In some instances, the thickness of a substrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or 250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some instances, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials are connected to an electric ground. In some instances, the substrate is heat conductive or insulated. In some instances, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example polynucleotide synthesis reaction processes. In some instances, a substrate comprises flexible materials. For flexible materials, materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. For rigid materials, materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); and metals (for example, gold, platinum, and the like). The substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures/reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some instances, a substrate comprises raised and/or lowered features. One benefit of having such features is an increase in surface area to support polynucleotide synthesis. In some instances, a substrate having raised and/or lowered features is referred to as a three-dimensional substrate. In some cases, a three-dimensional substrate comprises one or more channels. In some cases, one or more loci comprise a channel. In some cases, the channels are accessible to reagent deposition via a deposition device such as a material deposition device. In some cases, reagents and/or fluids collect in a larger well in fluid communication one or more channels. For example, a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster. In some methods, a library of polynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some instances, the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface. In some instances, the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide. In some instances, a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein. In some instances, segregation is achieved by physical structure. In some instances, segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis. In some instances, differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots. In some cases, a device, such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations. Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g., more than about 10,000) with a low error rate (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases, a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height, and/or volume as another well of the substrate. A channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some instances, the diameter of a cluster or the diameter of a well comprising a cluster, or both, is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some instances, the diameter of a cluster or well or both is less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some instances, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1.150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm. The diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100, 80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, to about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, to about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface, wherein the surface comprises various surface modifications. In some instances, the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface. For example, surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.

In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. In some cases, surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for polynucleotide synthesis, are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three-dimensional features). In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules, and the like.

In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Pat. No. 5,474,796, which is herein incorporated by reference in its entirety.

In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry. In some instances, polynucleotide synthesis comprises coupling a base with phosphoramidite. Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional. Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps. Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3′ to 5′ direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the device activated. In some instances, the nucleoside phosphoramidite is provided to the device with an activator. In some instances, nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the device is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole may react, to a small extent, with the O6 position of guanosine. Without being bound by theory, upon oxidation with I₂/water, this side product, possibly via O6-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The 06 modifications may be removed by treatment with the capping reagent prior to oxidation with I₂/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the device is optionally washed.

In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the device bound growing nucleic acid is oxidized. The oxidation step comprises a phosphite triester which is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the device and growing polynucleotide are optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5′ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.

Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides. The synthesis may be in parallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel. The total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250, 20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in the art appreciate that the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100. The total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range. Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciate that the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10,000; 30,000; 75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours, or less. In some instances, larger nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours, or less.

In some instances, methods described herein provide for generation of a library of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites. In some instances, a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent but are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.

Referring to the Figures, FIG. 3 illustrates an exemplary process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids. The workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.

Once large nucleic acids for generation are selected, a predetermined library of nucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density polynucleotide arrays. In the workflow example, a device surface layer is provided. In the example, chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.

In situ preparation of polynucleotide arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step-wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 302. In some instances, polynucleotides are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 303. Prior to or after the sealing 304 of the polynucleotides, a reagent is added to release the polynucleotides from the substrate. In the exemplary workflow, the polynucleotides are released subsequent to sealing of the nanoreactor 305. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long-range sequence of DNA. Partial hybridization 305 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerase cycles, the polynucleotides anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for formation of a complete large span of double stranded DNA 306.

After PCA is complete, the nanoreactor is separated from the device 307 and positioned for interaction with a device having primers for PCR 308. After sealing, the nanoreactor is subject to PCR 309 and the larger nucleic acids are amplified. After PCR 310, the nanochamber is opened 311, error correction reagents are added 312, the chamber is sealed 313 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 314. The nanoreactor is opened and separated 315. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 322 for shipment 323.

In some instances, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 316, sealing the wafer to a chamber containing error corrected amplification product 317, and performing an additional round of amplification 318. The nanoreactor is opened 319 and the products are pooled 320 and sequenced 321. After an acceptable quality control determination is made, the packaged product 322 is approved for shipment 323.

In some instances, a nucleic acid generated by a workflow such as that in FIG. 3 is subject to mutagenesis using overlapping primers disclosed herein. In some instances, a library of primers is generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 302.

Computer Systems

Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. In various instances, the methods and systems of the disclosure may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the disclosure. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.

The computer system 400 illustrated in FIG. 4 may be understood as a logical apparatus that can read instructions from media 411 and/or a network port 405, which can optionally be connected to server 409 having fixed media 412. The system, such as shown in FIG. 4 can include a CPU 401, disk drives 403, optional input devices such as keyboard 415 and/or mouse 416 and optional monitor 407. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 422 as illustrated in FIG. 4.

FIG. 5 is a block diagram illustrating a first example architecture of a computer system 500 that can be used in connection with example instances of the present disclosure. As depicted in FIG. 5, the example computer system can include a processor 502 for processing instructions. Non-limiting examples of processors include: Intel Xeonm processor, AMD Opteronm processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some instances, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.

As illustrated in FIG. 5, a high-speed cache 504 can be connected to, or incorporated in, the processor 502 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by the processor 502. The processor 502 is connected to a north bridge 506 by a processor bus 508. The north bridge 506 is connected to random access memory (RAM) 510 by a memory bus 512 and manages access to the RAM 510 by the processor 502. The north bridge 506 is also connected to a south bridge 514 by a chipset bus 516. The south bridge 514 is, in turn, connected to a peripheral bus 518. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 518. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some instances, system 500 can include an accelerator card 522 attached to the peripheral bus 518. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

Software and data are stored in external storage 524 and can be loaded into RAM 510 and/or cache 504 for use by the processor. The system 500 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example instances of the present disclosure. In this example, system 500 also includes network interface cards (NICs) 520 and 521 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.

FIG. 6 is a diagram showing a network 600 with a plurality of computer systems 602a, and 602b, a plurality of cell phones and personal data assistants 602c, and Network Attached Storage (NAS) 604a, and 604b. In example instances, systems 602a, 602b, and 602c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 604a and 604b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 602a, and 602b, and cell phone and personal data assistant systems 602c. Computer systems 602a, and 602b, and cell phone and personal data assistant systems 602c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 604a and 604b. FIG. 6 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various instances of the present disclosure. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface. In some example instances, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other instances, some or all of the processors can use a shared virtual address memory space.

FIG. 7 is a block diagram of a multiprocessor computer system 700 using a shared virtual address memory space in accordance with an example instance. The system includes a plurality of processors 702a-f that can access a shared memory subsystem 704. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 706a-f in the memory subsystem 704. Each MAP 706a-f can comprise a memory 708a-f and one or more field programmable gate arrays (FPGAs) 710a-f. The MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs 710a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example instances. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 708a-f, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor 702a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example instances, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some instances, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example instances, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other instances, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 5, system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 522 illustrated in FIG. 5.

The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H₂SO₄and 10% H₂O₂for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N2. The device was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The device was then plasma cleaned by exposing the device surface to 02. A SAMCO PC-300 instrument was used to plasma etch 02 at 250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O₂plasma etch at 250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with N2. The functionalized surface was activated to serve as a support for polynucleotide synthesis.

Example 2: Synthesis of a 50-mer Sequence on an Oligonucleotide Synthesis Device

A two-dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (AB1394 DNA Synthesizer”). The two-dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) which was used to synthesize an exemplary polynucleotide of 50 bp (“50-mer polynucleotide”) using polynucleotide synthesis methods described herein.

The sequence of the 50-mer was as described in SEQ ID NO: 1348. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQ ID NO.: 1348), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 3 and an ABI synthesizer.

TABLE 3

Synthesis protocols

General DNA Synthesis
Table 3

Process Name
Process Step
Time (sec)

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
Acetonitrile to Flowcell
23

N2 System Flush
4

Acetonitrile System Flush
4

DNA BASE ADDITION
Activator Manifold Flush
2

(Phosphoramidite +
Activator to Flowcell
6

Activator Flow)
Activator +
6

Phosphoramidite to

Flowcell

Activator to Flowcell
0.5

Activator +
5

Phosphoramidite to

Flowcell

Activator to Flowcell
0.5

Activator +
5

Phosphoramidite to

Flowcell

Activator to Flowcell
0.5

Activator +
5

Phosphoramidite to

Flowcell

Incubate for 25 sec
25

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
Acetonitrile to Flowcell
15

N2 System Flush
4

Acetonitrile System Flush
4

DNA BASE ADDITION
Activator Manifold Flush
2

(Phosphoramidite +
Activator to Flowcell
5

Activator Flow)
Activator +
18

Phosphoramidite to

Flowcell

Incubate for 25 sec
25

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
Acetonitrile to Flowcell
15

N2 System Flush
4

Acetonitrile System Flush
4

CAPPING (CapA + B, 1:1,
CapA + B to Flowcell
15

Flow)

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
Acetonitrile to Flowcell
15

Acetonitrile System Flush
4

OXIDATION (Oxidizer
Oxidizer to Flowcell
18

Flow)

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
N2 System Flush
4

Acetonitrile System Flush
4

Acetonitrile to Flowcell
15

Acetonitrile System Flush
4

Acetonitrile to Flowcell
15

N2 System Flush
4

Acetonitrile System Flush
4

Acetonitrile to Flowcell
23

N2 System Flush
4

Acetonitrile System Flush
4

DEBLOCKING (Deblock
Deblock to Flowcell
36

Flow)

WASH (Acetonitrile Wash
Acetonitrile System Flush
4

Flow)
N2 System Flush
4

Acetonitrile System Flush
4

Acetonitrile to Flowcell
18

N2 System Flush
4.13

Acetonitrile System Flush
4.13

Acetonitrile to Flowcell
15

The phosphoramidite/activator combination was delivered similarly to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an Oligonucleotide Synthesis Device

The same process as described in Example 2 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer polynucleotide (“100-mer polynucleotide”; 5′ CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATG CTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes); SEQ ID NO.: 1349) on two different silicon chips, the first one uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane, and the polynucleotides extracted from the surface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using a forward (5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 1350) and a reverse (5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO.: 1351) primer in a 50 uL PCR mix (25 uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverse primer, 1 uL polynucleotide extracted from the surface, and water up to 50 uL) using the following thermalcycling program:

- 98° C., 30 sec
- 98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles
- 72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 4 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 4

Sequencing results

Spot
Error rate
Cycle efficiency

1
1/763 bp
99.87%

2
1/824 bp
99.88%

3
1/780 bp
99.87%

4
1/429 bp
99.77%

5
1/1525 bp
99.93%

6
1/1615 bp
99.94%

7
1/531 bp
99.81%

8
1/1769 bp
99.94%

9
1/854 bp
99.88%

10
1/1451 bp
99.93%

Thus, the high quality and uniformity of the synthesized polynucleotides were repeated on two chips with different surface chemistries. Overall, 89% of the 100-mers that were sequenced were perfect sequences with no errors, corresponding to 233 out of 262.

Table 5 summarizes error characteristics for the sequences obtained from the polynucleotide samples from spots 1-10.

TABLE 5

Error characteristics

Sample ID/Spot no.

OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_
OSA_

0046/1
0047/2
0048/3
0049/4
0050/5
0051/6
0052/7
0053/8
0054/9
0055/10

Total
32
32
32
32
32
32
32
32
32
32

Sequences

Sequencin
25 of
27 of
26 of
21 of
25 of
29 of
27 of
29 of
28 of
25 of

g Quality
28
27
30
23
26
30
31
31
29
28

Oligo
23 of
25 of
22 of
18 of
24 of
25 of
22 of
28 of
26 of
20 of

Quality
25
27
26
21
25
29
27
29
28
25

ROI
2500
2698
2561
2122
2499
2666
2625
2899
2798
2348

Match

Count

ROI
2
2
1
3
1
0
2
1
2
1

Mutation

ROI Multi
0
0
0
0
0
0
0
0
0
0

Base

Deletion

ROI Small
1
0
0
0
0
0
0
0
0
0

Insertion

ROI
0
0
0
0
0
0
0
0
0
0

Single

Base

Deletion

Large
0
0
1
0
0
1
1
0
0
0

Deletion

Count

Mutation:
2
2
1
2
1
0
2
1
2
1

G > A

Mutation:
0
0
0
1
0
0
0
0
0
0

T > C

ROI Error
3
2
2
3
1
1
3
1
2
1

Count

ROI Error
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1
Err: ~1

Rate
in 834
in 1350
in 1282
in 708
in 2500
in 2667
in 876
in 2900
in 1400
in 2349

ROI
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:
MP Err:

Minus
~1 in
~1 in
~1 in
~1 in
~1 in
~1 in
~1 in
~1 in
~1 in
~1 in

Primer
763
824
780
429
1525
1615
531
1769
854
1451

Error Rate

Example 4: Functional GLP-1R Antibodies Identified from a Synthetic GPCR-Focused Library Demonstrate Potent Blood Glucose Control

This example describes the identification of antagonistic and agonistic GLP-1R antibodies with in vitro and in vivo functional activity.

Materials and Method

Stable Cell Line and Phage Library Generation

The full length human GLP-1R gene (UniProt—P43220) with an N-terminal FLAG tag and C-terminal GFP tag cloned into pCDNA3.1(+) vector (ThermoFisher) was transfected into suspension Chinese Hamster Ovary (CHO) cells to generate the stable cell line expressing GLP-1R. Target expression was confirmed by FACS. Cells expressing >80% of GLP-1R by GFP were then directly used for cell-based selections.

Germline heavy chain IGHV1-69, IGHV3-30 and germline light chain IGKV1-39, IGKV3-15, IGLV1-51, IGLV2-14 framework combinations were used in the GPCR-focused phage-displayed library, and all six CDR diversities were encoded by oligo pools synthesized similar to Examples 1-3 above. The CDRs were also screened to ensure they did not contain manufacturability liabilities, cryptic splice sites, or commonly used nucleotide restriction sites. The heavy chain variable region (VH) and light chain variable region (VL) were linked by (G4S)3 linker (SEQ ID NO: 1520). The resulting scFv (VH-linker-VL) gene library was cloned into a pADL 22-2c (Antibody Design Labs) phage display vector by NotI restriction digestion and electroporated into TG1 electro-competent E. coli cells. (Lucigen). The final library has a diversity of 1.1×10¹⁰size, which was verified by NGS.

Panning and Screening Strategy Used to Isolate Agonist GLP-1R scFv Clones

Before panning on GLP-1R expressing CHO cells, phage particles were blocked with 5% BSA/PBS and depleted for non-specific binders on CHO parent cells. For CHO parent cell depletion, the input phage aliquot was rotated at 14 rpm/min with 1×10⁸CHO parent cells for 1 hour at room temperature (RT). The cells were then pelleted by centrifuging at 1,200 rpm for 10 mins in a tabletop Eppendorf centrifuge 5920RS/4×1000 rotor to deplete the non-specific CHO cell binders. The phage supernatant, depleted of CHO cell binders, was then transferred to 1×10⁸GLP-1R expressing CHO cells. The phage supernatant and GLP-1R expressing CHO cells were rotated at 14 rpm/min for 1 hour at RT to select for GLP-1R binders. After incubation, the cells were washed several times with 1×PBS/0.5% Tween to remove non-binding clones. To elute the phage bound to the GLP-1R cells, the cells were incubated with trypsin in PBS buffer for 30 minutes at 37° C. The cells were pelleted by centrifuging at 1,200 rpm for 10 mins. The output supernatant enriched in GLP-1R binding clones was amplified in TG1 E. coli cells to use as input phage for the next round of selection. This selection strategy was repeated for five rounds. Every round was depleted against the CHO parent background. Amplified output phage from a round was used as the input phage for the subsequent round, and the stringency of washes were increased in each subsequent round of selections with more washes. After five rounds of selection, 500 clones from each of round 4 and round 5 were Sanger sequenced to identify unique clones.

Next-Generation Sequencing Analysis

The phagemid DNA was miniprepped from the output bacterial stocks of all panning rounds. The variable heavy chain (VH) was PCR amplified from the phagemid DNA using the Forward Primer ACAGAATTCATTAAAGAGGAGAAATTAACC (SEQ ID NO: 1521) and reverse primer TGAACCGCCTCCACCGCTAG (SEQ ID NO: 1522). The PCR product was directly used for library preparation using the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, product #KK8514). To add diversity in the library, the samples were spiked with 15% PhiX Control purchased from Illumina, Inc. (product #FC-110-3001). The library was then loaded onto Illumina's 600 cycle MiSeq Reagent Kit v3 (Illumina, product #MS-102-3003) and run on the MiSeq instrument.

Reformatting and High Throughput (HT) IgG Purification

Expi293 cells were transfected using Expifectamine (ThermoFisher, A14524) with the heavy chain and light chain DNA at a 2:1 ratio and supernatants were harvested 4 days post-transfection before cell viability dropped below 80%. Purifications were undertaken using either King Fisher (ThermoFisher) with protein A magnetic beads or Phynexus protein A column tips (Hamilton). For large scale production of IgG clones that were evaluated in in vivo mouse studies, an Akta HPLC purification system (GE) was used.

IgG characterization and quality control. The purified IgGs for the positive GLP-1R binders (hits) were subjected to characterization for their purity by LabChip GXII Touch HT Protein Express high-sensitivity assay. Dithiothreitol (DTT) was used to reduce the IgG into VH and VL. IgG concentrations were measured using Lunatic (UnChain). IgG for in vivo mouse studies were further characterized by HPLC and tested for endotoxin levels (Endosafe® nexgen-PTS™ Endotoxin Testing, Charles River), with less than 5 EU per kg dosing.

Binding Assays and Flow Cytometry

GLP-1R IgG clones were tested in a binding assay coupled to flow cytometry analysis as follows: FLAG-GLP-1R-GFP expressing CHO cells (CHO-GLP-1R) and CHO-parent cells were incubated with 100 nM IgG for 1 h on ice, washed three times and incubated with Alexa 647 conjugated goat-anti-human antibody (1:200) (Jackson ImmunoResearch Laboratories, 109-605-044) for 30 min on ice, followed by three washes, centrifuging to pellet the cells between each washing step. All incubations and washes were in buffer containing PBS+1% BSA. For titrations, IgG was serially diluted 1:3 starting from 100 nM down to 0.046 nM. Cells were analyzed by flow cytometry and hits (a hit is an IgG that specifically binds to CHO-GLP-1R) were identified by measuring the GFP signal against the Alexa 647 signal. Flow cytometry data of binding assays with 100 nM IgG are presented as dot plots. Analyses of binding assays with IgG titrations are presented as binding curves plotting IgG concentrations against MFI (mean fluorescence intensity).

Ligand Competition Assay

Ligand competition assays involved co-incubating the primary IgG with 1 μM GLP-1 (7-36). For each data point, IgG (600 nM) was prepared in Flow buffer (PBS+1% BSA) and diluted 1:3 down for 8 titration points. Peptide GLP-1 7-36 (2 μM) was prepared similarly with the Flow buffer (PBS+1% BSA). Each well contained 100,000 cells to which 50 μL of IgG and 50 μL of peptide (=plus) or buffer alone without peptide (=minus) were added. Cells and IgG/peptide mix were incubated for 1 hr on ice, and after washing, secondary antibody (goat anti-human APC, Jackson ImmunoResearch Laboratories, product #109-605-044) diluted 1:200 in PBS+1% BSA was added. This was incubated on ice for 30 mins (50 μL per well), before washing and resuspending in 60 μL buffer. Finally, the assay read-out was measured on an Intellicyt® IQue3 Screener at a rate of 4 seconds per well.

Cell-Based Functional Assays

cAMP assays. GLP-1R IgG clones were tested for their potential effects on GLP-1R signaling by performing cAMP assays obtained from Eurofins DiscoverX. The technology involved in detecting cAMP levels is a no wash gain-of-signal competitive immunoassay based on Enzyme Fragment Complementation technology. Experiments were designed to test for either agonist or antagonist activity of the IgG clones. To test for agonist activity of the IgGs, cells were stimulated with IgG incubating for 30 min at 37° C. (titrations 1:3 starting from 100 nM and diluting down to 0.046 nM with PBS) or with the known agonist GLP-1 7-36 peptide (MedChemExpress, Cat. No.: HY-P005), titrated 1:6 starting from 12.5 nM and diluting down to 0.003 nM with PBS. To test for antagonist activity, cells were incubated with IgG at a fixed concentration of 100 nM for 1 h at room temperature to allow binding, followed by stimulation with GLP1 7-36 peptide (titrations 1:6 starting from 12.5 nM down to 0.003 nM in PBS) for 30 min at 37° C. Intracellular cAMP levels were detected by following the assay kit instructions.

Beta arrestin recruitment assy. β-arrestin recruitment assay was obtained from Eurofins DiscoverX (Cat #93-0300E2) that utilized untagged GLP-1R overexpressing CHO-K1 cells. The experiment is to test if GLP1R-3 has an effect on GLP-1 7-36 agonist induced β-arrestin recruitment upon GLP-1R activation. Expanded cells were seeded into 96 well plates at 5,000 cells/well, and the experiment was performed 48 hours after plating cells. 100 nM IgG was pre-incubated for 1 hour at RT with plated cells in 50 ul volume, and then 5 ul of ligand GLP-1 7-36 was added for a further incubation for 30 min at 37° C. Add 22.5 uL of detection solution to each well, tap gently and briefly spin down. Then incubate plates at RT for 1 hour in the dark. The plates were then read by a Chemiluminescence plate reader, Molecular Devices SpectraMax M5, and output relative light units (RLU) data were analyzed using GraphPad Prism.

In Vivo Studies

Animals. All animal procedures were approved by Institutional Animal Care and Use Committee (IACUC) at the University of California San Francisco and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of laboratory Animals. C57BL/6NHsd (Envigo RMS, LLC) male littermates at 8-10 weeks of age, weighing ˜20-28 grams, were used in all the studies described. The mice were housed in a room that was temperature (22-25 C) and light controlled (12-h: 12-h light/dark cycle starting at 7 AM. The mice were fed with chow diet with 9% fat (PicoLab mouse Diet 20 (#5058), Lab Supply, Fortworth Tex., USA) for the duration of housing at the UCSF animal care facility.

Monoclonal Antibodies and Reagents. Anti-GLP-1 monoclonal antibodies (mAb) in PBS buffer were tested in these studies an agonist mAb, GLP1R-59-2 and one antagonist mAb, GLP1R-3. Mice were dosed prior to a Glucose Tolerance Test (GTT) or an Insulin Tolerance test (ITT) using the following regimen: Agonist GLP1R-59-2 mAb was dosed at 5 or 10 mg/kg at three different administration regimen groups prior to performing a GTT and with four different administration regimen groups in an Insulin Tolerance test (ITT). 1. mAb administered as a single dose, 15 hours prior to GTT and 21 hours prior to ITT. 2. mAb administered as a double dose, 15 hours prior to GTT and 21 hours prior to ITT plus a second mAb dose 2 hours prior to GTT and ITT. 3. mAb single dose 2 hours prior to GTT and ITT. 4. mAb single dose 6 hours prior to ITT only.

Antagonist GLP1R-3 mAb was dosed at 20 mg/kg at four different administration regimen groups. 1. mAb administered as a single dose, 15 hours prior to GTT and 21 hours prior to ITT. 2. mAb administered as a double dose, 15 hours prior to GTT and 21 hours prior to ITT plus a second mAb dose 2 hours prior to GTT and ITT. 3. mAb as a single dose 6 hours prior to GTT and ITT. 4. mAb single dose 2 hours prior to GTT and ITT.

Extendin 9-39 Peptide (MedChemExpress, Cat. No.: HY-P0264) were dosed at 1.0 or 0.23 mg/kg at three different administration regimen groups. 1. Extendin administered as a single dose, 21 hours prior to ITT. 2. Extendin administered as a double dose, 21 hours prior to ITT plus a second Extendin dose 2 hours prior to ITT. 3. mAb as a single dose 6 hours prior to ITT.

Glucose Tolerance Test

A Glucose Tolerance Test (GTT) was used to assess two different anti-GLP1 mAbs (Agonist and Antagonist) effect on glucose tolerance following an acute glucose administration. Intraperitoneal Glucose Tolerance Test (IP-GTT) was conducted in 8 or 10-week old male mice to assess glucose disposal after a glucose injection and measuring blood glucose level after mice were fasted overnight (14-16 hours). To avoid circadian variations in mouse blood glucose levels this testing was performed at fixed times. Mice were weighed after the overnight fast and baseline blood glucose levels (pre-glucose injection; Time 0 minutes) were measured. Mice were injected, i.p., with a single bolus (10 ul/gram body weight) of 30% Dextrose solution (Hospira, Ill.) and blood glucose levels were measured at 15, 30, 60, 120 and 180-minutes post glucose administration. Blood samples were obtained by a tail nick and blood glucose levels were monitored using a OneTouch Ultra 2 glucose monitor (LifeScan, Inc.)

Insulin Tolerance Test

An Insulin Tolerance Test (ITT) was conducted to assess two different anti-GLP1 mAbs (agonist and antagonist) effect on insulin sensitivity following acute insulin administration. 8 or 10-week old male mice were fasted for 6 hours and body weight was recorded before and after fasting. To avoid circadian variations in mouse blood glucose levels this testing was performed at fixed times. Blood samples were collected by tail nick and baseline glucose was measured prior to insulin injection. Mice were injected, i.p., with a single bolus (0.75 U/Kg body weight) of human insulin (Novolin, Novo Nordisk) and blood glucose levels were measured at 15, 30, 45, 60 and 120 minutes after insulin injection. Blood glucose levels were monitored using a OneTouch Ultra 2 glucose monitor (LifeScan, Inc.).

ELISA for Pharmacokinetic (PK) Studies.

The rat PK study was done at Charles River Laboratories, One Innovation Dr, 3 Biotech, Worcester, Mass. 01605. 5 Male Sprague-Dawley rats per group were allowed to acclimate after receiving at test facility for a minimum of 3 days before dosing. GLP1R-3 and GLP1R59-2 were dosed at 10 mg/kg by IV in 100 mM Hepes, 100 mM NaCl, 50 mM NaAc, pH 6.0 vehicle. Serial blood samples were collected via jugular vein cannula with ˜250 ul volume at each time point: pre-dose, 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 168, 240 and 336 hours post dose. Blood samples were collected into K₂EDTA tubes and stored on wet ice until processed to plasma by centrifugation (3500 rpm for 10 minutes at 5° C.) within 30 minutes of collection. Plasma samples were then transferred into an appropriate tube containing DPP-4 (3.3 μL for 100 μL of plasma) and frozen on dry ice. To measure the human IgG in rat plasma samples, sheep anti-Human IgG (1 mg/mL) was used as coating reagent) (The binding site, Lot No. AU003.M), and goat anti-Human IgG, HRP (H&L) (1 mg/mL) was used as detection reagent) (Bethyl, cat #A80-319P) in an ELISA assay. Stock solutions of human IgG standards and QCs were prepared by spiking human IgG into rat plasma. A minimum of two wells were used to analyze each study samples, QC's, standards, and blank. A 4-parameter logistic (4PL) model was used to fit the sigmoid calibration curve. The semi-logarithmic sigmoid calibration curve was obtained by plotting the absorbance response against concentration. Concentrations of analyte in the test samples were determined by computer interpolation from the plot of the calibration curve.

Results

Design of GPCR-Focused Antibody Library is Based on GPCR Binding Motifs and GPCR Antibodies

All known GPCR interactions, which include interactions of GPCRs with ligands, peptides, antibodies, endogenous extracellular loops and small molecules were analyzed to map the GPCR binding molecular determinants. Crystal structures of almost 150 peptides, ligand or antibodies bound to ECDs of around 50 GPCRs (http://www.gpcrdb.org) were used to identify GPCR binding motifs. Over 1000 GPCR binding motifs were extracted from this analysis. In addition, by analysis of all solved structures of GPCRs (zhanglab.ccmb.med.umich.edu/GPCR-EXP/), over 2000 binding motifs from endogenous extracellular loops of GPCRs were identified. Finally, by analysis of structures of over 100 small molecule ligands bound to GPCR, a reduced amino acid library of 5 amino acids (Tyr, Phe, His, Pro and Gly) that may be able to recapitulate many of the structural contacts of these ligands was identified. A sub-library with this reduced amino acid diversity was placed within a CxxxxxC motif. In total, over 5000 GPCR binding motifs were identified (FIGS. 9A-9E). These binding motifs were placed in one of five different stem regions:

(SEQ ID NO: 1523)

CARDLRELECEEWTxxxxxSRGPCVDPRGVAGSFDVW,

(SEQ ID NO: 1524)

CARDMYYDFxxxxxEVVPADDAFDIW,

(SEQ ID NO: 1525)

CARDGRGSLPRPKGGPxxxxxYDSSEDSGGAFDIW,

(SEQ ID NO: 1526)

CARANQHFxxxxxGYHYYGMDVW,

(SEQ ID NO: 1527)

CAKHMSMQxxxxxRADLVGDAFDVW.

These stem regions were selected from structural antibodies with ultra-long HCDR3s. Antibody germlines were specifically chosen to tolerate these ultra-long HCDR3s. Structure and sequence analysis of human antibodies with longer than 21 amino acids revealed a V-gene bias in antibodies with long CDR3s. Finally, the germline IGHV (IGHV1-69 and IGHV3-30), IGKV (IGKV1-39 and IGKV3-15) and IGLV (IGLV1-51 and IGLV2-14) genes were chosen based on this analysis.

In addition to HCDR3 diversity, limited diversity was also introduced in the other 5 CDRs. There were 416 HCDR1 and 258 HCDR2 variants in the IGHV1-69 domain; 535 HCDR1 and 416 HCDR2 variants in the IGHV3-30 domain; 490 LCDR1, 420 LCDR2 and 824 LCDR3 variants in the IGKV1-39 domain; 490 LCDR1, 265 LCDR2 and 907 LCDR3 variants in the IGKV3-15 domain; 184 LCDR1, 151 LCDR2 and 824 LCDR3 variants in the IGLV1-51 domain; 967 LCDR1, 535 LCDR2 and 922 LCDR3 variants in the IGLV2-14 domain (FIG. 10). These CDR variants were selected by comparing the germline CDRs with the near-germline space of single, double and triple mutations observed in the CDRs within the V-gene repertoire of at least two out of 12 human donors. All CDRs have were pre-screened to remove manufacturability liabilities, cryptic splice sites or nucleotide restriction sites. The CDRs were synthesized as an oligo pool and incorporated into the selected antibody scaffolds. The heavy chain (VH) and light chain (VL) genes were linked by (G₄S)₃linker (SEQ ID NO: 1520). The resulting scFv (VH-linker-VL) gene pool was cloned into a phagemid display vector at the N-terminal of the M13 gene-3 minor coat protein. The final size of the GPCR library is 1×10¹⁰in a scFv format. Next-generation sequencing (NGS) was performed on the final phage library to analyze the HCDR3 length distribution in the library for comparison with the HCDR3 length distribution in B-cell populations from three healthy adult donors. The HCDR3 sequences from the three healthy donors used were derived from a publicly available database with over 37 million B-cell receptor sequences³¹. The HCDR3 length in the GPCR library is much longer than the HCDR3 length observed in B-cell repertoire sequences. On average, the median HCDR3 length in the GPCR library (which shows a biphasic pattern of distribution) is two or three times longer (33 to 44 amino acids) than the median lengths observed in natural B-cell repertoire sequences (15 to 17 amino acids) (FIG. 11). The biphasic length distribution of HCDR3 in the GPCR library is mainly caused by the two groups of stems (8aa, 9aaxxxxx10aa, 12aa) and (14aa, 16aa xxxxx18aa, 14aa) used to present the motifs within HCDR3.

Phage Panning Against GLP-1R Over-Expressing Cell Lines Resulted in Clonal Enrichment

A GLP-1R over-expressing CHO stable cell line was created with a FLAG tag presented on the N-terminus of the receptor in order to detect cell surface expression and an EGFP tag on the C-terminus to track total receptor expression. Flow cytometry analysis of these cells confirmed that the majority of the receptor (>80%) was expressed at the cell surface (FIG. 12A). These GLP-1R-expressing CHO cells were used for five rounds of phage panning against the GPCR-focused library. The selection scheme is outlined in FIG. 12B. The variable heavy chain (VH) from the output of each panning round was PCR amplified and sequenced by MiSeq. As the percent unique HCDR3 decreases in each round output pool NGS sequencing, significant clonal enrichment was observed from round 1 to round 5 (FIG. 13), indicating a target specific clonal selection in the panning process. Approx. 1000 clones in total (from round 4 and round 5) were picked for single clonal NGS sequencing and ˜100 unique VH-VL pairs were selected to be reformatted and expressed as full length human IgG2 at 1 ml scale.

IgG Binders Directed to GLP-1R Contain Either GLP-1, GLP-2 or Unique HCDR3 Motifs Identified

Purified IgG clones were tested for specific binding to GLP-1R-expressing CHO cells. A single-point flow cytometry analysis using 100 nM of IgG concentration revealed that out of 100 IgG unique clones tested, 13 IgG clones bound specifically to GLP-1R-positive cells (GFP+) and not parental CHO cells (GFP−). The binding of these 13 hits was then further evaluated by 8-point titrations of each IgG clone starting from 200 nM (30 μg/mL) and the cell binding affinities were determined to be in the double-digit nM range. The average CHO parental cell background binding by all 13 IgG clones is shown as a black line and is minimal compared with specific binding to GLP-1R-expressing cells (FIG. 14). Full saturation was not observed, the plateau of the binding curve at the highest concentration, 200 nM used in the experiment. FIG. 15 shows the HCDR3 amino acid sequences of these 13 IgG clones. Six of these were found to include a GLP-1 motif, four included a GLP-2 motif, and three had unknown motif.

Eight IgGs of the 13 Binders are Negative Antagonists in GLP-1R Mediated cAMP Signaling

The 13 IgG binders were next assessed for their functional activity in the cAMP signaling pathway by using GLP-1R over-expressing CHO-K1 cells purchased from DiscoverX that are designed and validated for assessing GLP-1R-induced cAMP signaling. In the first instance, the IgG clones were tested for agonist activity as compared with the peptide agonist GLP-1 7-36 in dose titrations. While GLP-1 7-36 stimulation resulted in a cAMP signal, none was observed for the IgG clones, indicating that they are not activating. Subsequently, the panel of IgG clones were tested for antagonist activity by pre-incubating GLP-1R-expressing cells with a fixed concentration of IgG to allow binding to occur and then stimulating the cells with GLP-1 7-36 in a dose dependent manner. This allowed examination of the impact of the presence of IgG on GLP-1 7-36-induced GLP-1R cAMP signaling, thereby potentially revealing any potential competitive effects of the IgG. It was observed that the GLP-1 7-36 dose response curve shifted to the right in the presence of 8 out of the 13 IgG clones, suggesting that they act as negative antagonists of the GLP-1 7-36 response (data not shown). Similar observations were made regarding the effect of the 13 IgG clones on Exendin-4 induced GLP-1R cAMP signaling response (data not shown). The remaining five IgG clones appeared to have no significant effects on GLP-1R cAMP signaling (data not shown).

Characterization of Mechanisms of Action of the Antagonist IgG GLP1R-3

To determine the mechanism of action of these resulting functional hits, subsequent studies focused one of the GLP-1 motif-containing IgG clones that demonstrated high binding affinity, as well as functionality: GLP1R-3. Ligand competition binding assays, the IgG effects on the GLP-1 dose response in cAMP signaling, and beta-arrestin recruitment assays were conducted, resulting in characterization of GLP1R-3 as follows:

Competition with the endogenous ligand in GLP-1R binding assays. To determine if GLP1R-3 binds to the orthosteric site on the receptor, N-terminal FLAG-tagged and C terminal GFP-tagged GLP-1R over-expressing CHO cells were incubated with a dose titration of GLP1R-3 starting at 100 nM in the presence or absence of a fixed concentration of the peptide agonist GLP-1 7-36 (1 μM). Flow cytometry analysis revealed significantly reduced binding of GLP1R-3 to GLP-1R (GFP+) in the presence of GLP-1 7-36. Whilst the presence of GLP-1 7-36 peptide does not completely ablate GLP1R-3 binding, this observation suggests that the antibody may bind to an overlapping epitope, or GLP1R-3 have stronger binding affinity for GLP-1 7-36 to compete for binding. (FIG. 16A).

GLP1R-3 antagonizes GLP-1 activated cAMP signaling. The next step was to determine if GLP1R-3 exhibits competitive antagonism for GLP-1R in a dose-dependent manner. GLP-1 7-36-induced cAMP signaling was examined in the presence of a constant concentration (100 nM) of GLP1R-3 with a dose titration of GLP-1 7-36 starting at 20 nM with a 3-fold down titration, and a clear dose-dependent inhibition of the cAMP signal was observed. The EC50 for GLP-1 7-36 peptide is 0.025 nM without presence of GLP1R-3, and 0.11 nM in the presence of 100 nM GLP1R-3 (FIG. 16B), supporting that GLP1R-3 is a competitive antagonist.

GLP1R-3 reduces β-arrestin recruitment upon GLP-1R activation. When a GPCR is activated by an agonist, β-arrestins are recruited to the GPCR from the cytosol, thereby excluding the receptor from further G protein interactions and leading to signal arrest, hence the name “arrestin”. To determine if GLP1R-3 had any effects on β-arrestin recruitment by activated GLP-1R, GLP-1R over-expressing CHO-K1 cells (DiscoverX) that are specifically designed and validated for assessing GLP-1R β-arrestin recruitment were employed in the following manner. Cells were pre-incubated with a fixed concentration of GLP1R-3 (100 nM) for 1 hr at room temperature to allow binding to occur and then stimulated with GLP-1 7-36. GLP1R-3, showed inhibition of GLP-1 7-36 peptide-induced beta arrestin recruitment to GLP-1R as evidenced by the right shift of GLP-1 7-36 dose response curve for β-arrestin recruitment (FIG. 16C). This indicated that GLP1R-3 reduces β-arrestin recruitment to GLP-1R, which is consistent with the observed reduced receptor activation. Thus, these cell-based assays indicate that GLP1R-3 is a competitive antagonist to GLP-1 7-36 for GLP-1R.

Design and Characterization of a GLP-1R Agonist IgG GLP1R-59-2

Since none of the 13 IgG hits showed any agonist activity, a GLP-1R agonist antibody (GLP1R-59-2) by linking the native GLP-1 7-36 peptide to the light chain N-terminal of a functionally inactive but GLP-1R-specific binder GLP1R-2 (FIG. 17) was engineered. GLP-1R binding assays, cAMP assays, and β-arrestin recruitment assays were conducted, resulting in characterization of GLP1R-59-2 as described here:

GLP1R-59-2 specifically binds to GLP-1R-expressing CHO cells. Flow cytometry analysis revealed that GLP1R-59-2 bound specifically to GLP-1R-positive cells (GFP+) and not parental CHO cells (GFP−), specific binding was also confirmed by GLP1R-59-2 dose titrations producing an apparent binding EC₅₀of 15.5 nM (FIG. 18A).

GLP1R-59-2 induces a GLP-1R cAMP response similar to GLP-1 7-36 GLP1R-59-2 was tested for agonist activity as compared with GLP-1 7-36 for stimulating GLP-1R over-expressing CHO-K1 cells (DiscoverX) with separate dose titration analyses conducted for both ligand and antibody. It was found that both induced similar cAMP signaling profile and their dose response curves had almost overlapping EC₅₀values, 0.042 nM for GLP1R-59-2 and 0.085 nM for GLP-1 7-36. (FIG. 18B) supporting the hypothesis that GLP1R-59-2 can act as an effective agonist for GLP-1R.

GLP1R-59-2 is less efficacious for β-arrestin recruitment to GLP-1R than GLP-1 7-36 To determine if GLP1R-59-2 was able to induce a similar level of β-arrestin recruitment to GLP-1R as GLP-1 7-36, GLP-1R over-expressing CHO-K1 cells (DiscoverX) were stimulated with dose titrations of each. It was found that less β-arrestin recruitment occurred with GLP1R-59-2 stimulation than with GLP-1 7-36 stimulation (FIG. 18C). Whilst GLP1R-59-2 is less efficacious than GLP-1 7-36 for the maximal β-arrestin recruitment, it would appear that the agonist IgG is slightly more potent with an EC₅₀of 0.042 nM, and 0.085 nM for GLP-1 7-36, respectively.

In Vivo PK and PD Testing of GLP1R-3 and GLP1R-59-2

Endogenous GLP-1 peptide has a very short serum half-life of only a few minutes, however GLP-1R antibodies can have significantly longer half-lives. This can be a considerable advantage over the current GLP-1 peptide analog therapeutics. An in vivo PK rat study was performed to evaluate the half-life of the antagonist GLP1R-3 and agonist GLP1R-59-2 in IgG format. In a 2-week PK study, GLP1R-3 exhibited an antibody-like in vivo half-life of ˜1-week in rats, while the agonist GLP-1 peptide-antibody fusion, GLP1R-59-2 exhibited >2-day half-life in rats (FIGS. 19A-19B). Liraglutide, the approved GLP-1R agonist for the treatment of Type II diabetes has a 13-hour half-life.

Agonist GLP1R-59-2 was tested for it's in vivo pharmacodynamic (PD) effects in Glucose tolerance test (GTT) using wild-type C57BL/6NHsd mouse model, in comparison with the vehicle control. Agonist mAb GLP1R-59-2 treatment, either dose (5 mg/kg and 10 mg/kg) or dosing regimen (2 hrs, 13+2 hrs, and 15 hrs before glucose challenge), significantly stabilized blood glucose even after a glucose challenge (FIG. 20A). Compared to control mice GLP1R-59-2 treatments are all significant (p<0.001) at reducing Area Under the Curve (AUC) in an GTT (FIG. 20B). However, there is no significant difference between each individual treatment timing or dose.

Antagonist, GLP1R-3 mAb and GLP-1 peptide Exendin 9-39 treatment, with 19+2 hours dosing regimen before insulin challenge, significantly stabilizes a higher blood glucose in wild-type C57BL/6NHsd mice (FIG. 21A). Compared to control mice GLP1R-3 mAb (20 mg/kg) and Exendin (1 mg/kg) treatments are both significant (p<0.0001) at stabilizing Area Under the Curve (AUC) in an ITT (FIG. 21B). However, there is no significant difference between GLP1R-3 and Control vs. Exendin (0.23 mg/kg) with 19+2 hour treatment.

Another experiment using a single 6 hour dosing regimen, antagonist, GLP1R-3 mAb treatment also significantly stabilizes a higher blood glucose after an insulin challenge compared to GLP-1 peptide Exendin 9-39 (1.0 or 0.23 mg/kg dose) or control (FIG. 22A). Compared to control mice, GLP1R-3 mAb (20 mg/kg) treatment at 6 hours, significantly (p<0.05) stabilizes Area Under the Curve (AUC) in an ITT. However, there is no significant difference between Control vs. Exendin (1.0 and 0.23 mg/kg) with the single 6 hour treatment (FIG. 22B).

GLP1R-3 mAb treatment was also compared to a comparator antibody GLP1R-226-1 and GLP1R-226-2. GLP1R-3 mAb treatment in a single 6 hour dosing regimen significantly stabilized a higher blood glucose after an insulin challenge (at time 0) compared to GLP1R-226-1 (20 mg/kg) or control (FIGS. 23A-23B). Compared to control mice, GLP1R-3 mAb (20 mg/kg) treatment at 6 hours, significantly (p<0.05) stabilized Area Under the Curve (AUC) in an ITT. There was no significant difference (p<0.05) between control vs. GLP1R-226-1 or GLP1R-226-2 with a single 6 hour treatment.

Example 5: GLP1R Variants

GLP1R-3 was optimized to generate additional GLP1R variants.

The panning strategy for GLP1R-221 and GLP1R-222 variants is seen in FIGS. 24A-24B. 768 clones from Round 4 and Round 5 were picked and sequenced on Miseq. 95 unique clones were reformatted. Data for GLP1R-221 and GLP1R-222 variants is seen in Tables 6A-6H. Sequences for the GLP1R-221 and GLP1R-222 variants are seen in Tables 9-13.

TABLE 6A

IgG
MFI Ratio
Subtraction

GLP1R-3
993.31197
232201

GLP1R-
914.54027
272235

221-065

GLP1R-
1174.8495
241813

221-075

GLP1R-
1484.8457
240383

221-017

GLP1R-
1015.9153
239520

221-033

GLP1R-
746.61867
235615.5

221-076

GLP1R-
711.73926
231701

221-092

GLP1R-
711.15764
222989.5

221-034

GLP1R-
927.53542
222368.5

221-066

GLP1R-
1067.8986
220848

221-084

GLP1R-
1119.868
220417

221-009

TABLE 6B

IgG
MFI Ratio
Subtraction

GLP1R-3
740.2
223614

GLP1R-222-
13.70825851
350309.5

052

GLP1R-222-
773.9745223
242714

016

GLP1R-222-
777.8080645
240810.5

023

GLP1R-222-
794.2474916
237181

014

GLP1R-222-
525.349537
226519

090

GLP1R-222-
983.9519651
225096

073

GLP1R-222-
774.5748709
224723.5

012

GLP1R-222-
711.0952381
223680

082

GLP1R-222-
850.1807692
220787

081

GLP1R-222-
946.2456522
217406.5

056

TABLE 6C

Median RL1-H of
Median RL1-H of

Sample
Expressing Singlets
Parent Singlets
MFI Ratio

GLP1R221-017
240545
162
1484.8

GLP1R221-075
242019
206
1174.8

GLP1R221-009
220614
197
1119.9

GLP1R221-084
221055
207
1067.9

GLP1R221-044
217533.5
209
1040.8

GLP1R221-033
239756
236
1015.9

GLP1R01-3
232435
234
993.3

GLP1R221-014
200638
203
988.4

GLP1R221-083
212185
215
986.9

GLP1R221-043
195703
201
973.6

GLP1R221-082
195548
202
968.1

GLP1R221-018
160183
167
959.2

GLP1R221-001
200655
213
942.0

GLP1R221-066
222608.5
240
927.5

GLP1R221-065
272533
298
914.5

GLP1R221-051
212862.5
234
909.7

GLP1R221-003
203683.5
226
901.3

GLP1R221-019
197108
224
879.9

GLP1R221-088
197424
225.5
875.5

GLP1R221-020
175621
205
856.7

GLP1R221-021
163480.5
192
851.5

GLP1R221-077
197424
236
836.5

GLP1R221-069
191848
230
834.1

GLP1R221-002
181529
219
828.9

GLP1R221-040
208274
251.5
828.1

GLP1R221-027
197258.5
241
818.5

GLP1R221-094
203152
253
803.0

GLP1R221-042
214005.5
268
798.5

GLP1R221-022
199293
252
790.8

GLP1R221-012
217522
283
768.6

GLP1R221-031
168691
221
763.3

GLP1R221-079
195512.5
257
760.7

GLP1R221-059
194935.5
257
758.5

GLP1R221-086
173390.5
229.5
755.5

GLP1R221-076
235931.5
316
746.6

GLP1R221-016
162165.5
220.5
735.4

GLP1R221-054
163917
224
731.8

GLP1R221-036
191269
264
724.5

GLP1R221-072
218347
303
720.6

GLP1R221-038
178492
248
719.7

GLP1R221-092
232027
326
711.7

GLP1R221-034
223303.5
314
711.2

GLP1R221-058
168846
240
703.5

GLP1R221-057
185403
268.5
690.5

GLP1R221-090
183560
268
684.9

GLP1R221-063
184038
274
671.7

GLP1R221-029
197088
305
646.2

GLP1R221-013
171640
266
645.3

GLP1R221-030
160279
251
638.6

GLP1R221-011
175641
283
620.6

GLP1R221-060
178266.5
290
614.7

GLP1R221-039
132161.5
219
603.5

GLP1R221-015
176341.5
293
601.8

GLP1R221-091
174624
295
591.9

GLP1R221-074
173151
295.5
586.0

GLP1R221-035
184526
315
585.8

GLP1R221-041
101875
174
585.5

GLP1R221-028
158490.5
271.5
583.8

GLP1R221-046
137324.5
236
581.9

GLP1R221-052
205979
370
556.7

GLP1R221-073
102371
205
499.4

GLP1R221-053
146049.5
301.5
484.4

GLP1R221-056
197814
409
483.7

GLP1R221-005
105542
226.5
466.0

GLP1R221-087
178772
389
459.6

GLP1R221-089
148048
325
455.5

GLP1R221-071
138673
313
443.0

GLP1R221-025
100871
233
432.9

GLP1R221-032
172291
399
431.8

GLP1R221-055
137657
329
418.4

GLP1R221-010
107233
285
376.3

GLP1R221-078
108233.5
301.5
359.0

GLP1R221-024
79574
225
353.7

GLP1R221-050
65939
204
323.2

GLP1R221-008
74751.5
239
312.8

GLP1R221-007
94850
358
264.9

GLP1R221-062
59544
279
213.4

GLP1R221-093
94190
444
212.1

GLP1R221-068
56581
298
189.9

GLP1R221-067
54810
300
182.7

GLP1R221-085
201695
1352.5
149.1

GLP1R221-064
42803
308
139.0

GLP1R221-023
155330
1174
132.3

GLP1R221-080
196473
1547
127.0

GLP1R221-061
47559
482
98.7

GLP1R221-070
21104.5
224
94.2

GLP1R221-006
17593.5
286
61.5

GLP1R221-045
603.5
174
3.5

GLP1R221-004
519
164
3.2

GLP1R221-047
397
167
2.4

GLP1R221-048
214
142.5
1.5

Stained Control
145
142
1.0

TABLE 6D

Median RL1-H of
Median RL1-H of

Sample
Expressing Singlets
Parent Singlets
MFI Ratio

GLP1R222-005
203990
173
1179.1

GLP1R222-058
217592
186
1169.8

GLP1R222-004
201104
189
1064.0

GLP1R222-035
180903
172
1051.8

GLP1R222-069
193190
187
1033.1

GLP1R222-001
195159
193
1011.2

GLP1R222-077
207327.5
208
996.8

GLP1R222-072
196881.5
198.5
991.8

GLP1R222-062
207390
209.5
989.9

GLP1R222-073
225325
229
984.0

GLP1R222-009
173411
176.5
982.5

GLP1R222-064
207016
218
949.6

GLP1R222-056
217636.5
230
946.2

GLP1R222-089
196242
213
921.3

GLP1R222-055
190727
209
912.6

GLP1R222-046
204177
225.5
905.4

GLP1R222-008
210228
234
898.4

GLP1R222-078
176537.5
198
891.6

GLP1R222-092
212558
240.5
883.8

GLP1R222-007
211051
239
883.1

GLP1R222-010
171471
195
879.3

GLP1R222-081
221047
260
850.2

GLP1R222-006
191343
227
842.9

GLP1R222-066
189419
227
834.4

GLP1R222-079
170284
206
826.6

GLP1R222-042
214181
261
820.6

GLP1R222-036
172934
214.5
806.2

GLP1R222-014
237480
299
794.2

GLP1R222-087
200143
252
794.2

GLP1R222-086
181615.5
230
789.6

GLP1R222-033
181334
230
788.4

GLP1R222-074
205325
261
786.7

GLP1R222-070
166040
212
783.2

GLP1R222-002
192431
246
782.2

GLP1R222-023
241120.5
310
777.8

GLP1R222-012
225014
290.5
774.6

GLP1R222-016
243028
314
774.0

GLP1R222-063
214679.5
278
772.2

GLP1R222-011
185538
242
766.7

GLP1R222-028
182568
242
754.4

GLP1R222-085
177368
239
742.1

GLP1R01-3
223916.5
302.5
740.2

GLP1R222-045
179811
246
730.9

GLP1R222-054
153121
211
725.7

GLP1R222-083
195648.5
274.5
712.7

GLP1R222-082
223995
315
711.1

GLP1R222-084
172287
247
697.5

GLP1R222-076
186158
269
692.0

GLP1R222-029
204757
300
682.5

GLP1R222-060
113206.5
167
677.9

GLP1R222-038
158998.5
236
673.7

GLP1R222-026
154255.5
229
673.6

GLP1R222-071
193867
288
673.1

GLP1R222-053
131845
196
672.7

GLP1R222-051
149756.5
224
668.6

GLP1R222-093
152427
232
657.0

GLP1R222-075
194948.5
297
656.4

GLP1R222-065
184054.5
281
655.0

GLP1R222-032
165221
255
647.9

GLP1R222-059
142048
223
637.0

GLP1R222-021
175543
278
631.4

GLP1R222-025
134869
216
624.4

GLP1R222-024
208523
345
604.4

GLP1R222-022
200898
337
596.1

GLP1R222-027
190430
326.5
583.2

GLP1R222-015
187125
344.5
543.2

GLP1R222-041
182770
344
531.3

GLP1R222-090
226951
432
525.3

GLP1R222-044
107845.5
208
518.5

GLP1R222-040
167413.5
324
516.7

GLP1R222-031
155641
331
470.2

GLP1R222-088
170891
373
458.2

GLP1R222-048
197618
441.5
447.6

GLP1R222-018
126619
290
436.6

GLP1R222-003
65950
155
425.5

GLP1R222-080
96756.5
228
424.4

GLP1R222-057
83288.5
204
408.3

GLP1R222-047
118739
307
386.8

GLP1R222-030
162896
506
321.9

GLP1R222-091
56735.5
192
295.5

GLP1R222-043
70814
406
174.4

GLP1R222-037
58889
388
151.8

GLP1R222-094
23462.5
176
133.3

GLP1R222-068
135253
1167.5
115.8

GLP1R222-019
39294
350
112.3

GLP1R222-067
146186
1452
100.7

GLP1R222-020
112537
1189
94.6

GLP1R222-049
178616.5
2138.5
83.5

GLP1R222-052
377875
27565.5
13.7

Stained Control
127
121
1.0

TABLE 6E

Sample

GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1

R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
GLP1

009
017
033
034
065
066
075
076
084
092
R01-3

EC₅₀
12.46
27.65
9.041
ND
ND
57.39
ND
ND
4.091
13.29
11.51

[nM]

CHO

GLP1R

B_max
215146
249646
167203
932518
797529
171812
213495
799149
286814
144511
179967

CHO

GLP1R

EC₅₀
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

[nM]

CHO

Parent

B_max

CHO
267.4
228.7
146
279.8
261.9
112.1
234
183.2
266.6
291.2
268

Parent

TABLE 6F

GLP
GLP1
GLP1
GLP1
GLP
GLP1
GLP1
GLP1
GLP
GLP1

1R222-
R222-
R222-
R222-
1R22
R222-
R222-
R222-
1R22
R222-
GLP1

Sample
012
014
016
023
2-052
056
073
081
2-082
090
R01-3

EC₅₀
23.14
34.29
7.709
18.35
17.36
77.43
13.07
22.51
11.49
ND
15.4

[nM]

CHO

GLP1R

B_max
233768
213081
129918
220325
228012
292619
150681
193955
134940
1078076
152782

CHO

GLP1R

EC₅₀
ND
ND
ND
89.37
ND
ND
ND
ND
ND
ND
ND

[nM]

CHO

Parent

B_max
340.6
336.4
218.5
237.9
47529
237.5
228.4
243.4
305
413.4
265.3

CHO

Parent

TABLE 6G

GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1

[IgG]
R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
R221-
GLP1

nM
009
017
033
034
065
066
075
076
084
092
R01-3

100.00
1635.4
1844.6
1596.5
1015.0
1157.8
1056.4
834.9
1499.3
910.9
960.9
1193.7

33.33
1322.9
1303.9
1211.3
593.5
799.1
698.8
507.9
597.8
666.7
1019.4
1531.0

11.11
1058.6
707.5
1012.5
332.2
368.9
229.7
416.1
372.2
412.4
447.3
689.3

3.70
448.3
424.8
385.6
209.0
280.0
171.2
242.0
293.6
344.2
297.4
425.2

1.23
176.6
181.4
175.6
87.7
140.1
91.4
119.1
121.3
153.3
141.2
166.6

0.41
95.2
94.7
89.7
48.9
80.0
46.5
54.7
51.8
63.5
54.9
77.4

0.14
37.7
36.2
39.3
19.7
31.0
20.4
23.8
22.3
24.6
19.6
28.6

0.05
16.8
14.8
17.4
8.8
14.9
9.6
9.3
8.8
9.4
8.7
12.3

TABLE 6H

GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1
GLP1

[IgG]
R222-
R222-
R222-
R222-
R222-
R222-
R222-
R222-
R222-
R222-
GLP1

nM
012
014
016
023
052
056
073
081
082
090
R01-3

100.00
1281.5
952.5
1049.1
1804.8
8.0
1522.2
1264.2
1404.0
845.8
746.6
1047.7

33.33
916.5
913.2
1412.1
1277.6
19.8
815.1
1057.9
1181.5
1027.4
526.8
1040.9

11.11
626.0
432.9
743.0
699.7
57.9
421.2
680.4
528.8
567.7
336.3
567.3

3.70
300.5
190.8
335.9
300.6
37.6
193.8
296.5
244.0
233.4
165.8
265.1

1.23
144.0
85.2
154.9
140.3
43.8
79.0
115.5
99.2
125.3
70.6
124.6

0.41
67.4
45.3
75.9
55.8
28.7
32.8
55.6
50.4
53.5
31.6
66.6

0.14
26.1
17.3
28.1
26.4
14.5
13.2
20.5
16.5
15.8
8.8
22.9

0.05
12.3
7.2
14.2
11.4
7.3
6.4
9.2
7.9
8.1
4.4
10.1

The GLP1R-221 and GLP1R-222 variants were assayed in competition assays. Data is seen in FIGS. 25A-25B. The variants were also assayed in a cAMP assay. Briefly, cells were pre-incubated with anti-GLP1R antibody at 100 nM followed by agonist stimulation 3× titration from 12.5 nM. Data is seen in FIG. 26 with improved variants highlighted in green.

Example 6: Sequences

TABLE 7

Sequences of GLP1 embedded in CDRH3

SEQ ID NO
Sequence

1
CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

2
CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW

3
CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW

4
CAKHMSMQEGAVTGEGQDAKEFIAWLVKGRVRADLVGDAFDVW

5
WAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

6
CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

7
CARANQHFYEQEGTFTSDVSSYLEGQAAKEFIAWLVKGGIRGYHYYGMDVW

8
CARANQHFTELHGEGQAAKEFIAWLVKGRGQIDIGYHYYGMDVW

9
CARANQHFLGAGVSSYLEGQAAKEFIAWLVKGDTTGYHYYGMDVW

10
CARANQHFLDKGTFTSDVSSYLEGQAAKEFIAWLVKGIYPGYHYYGMDVW

11
CARANQHFGTLSAGEGQAAKEFIAWLVKGGSQYDSSEDSGGAFDIW

12
CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW

13
CARANQHFGGKGEGQAAKEFIAWLVKGGGSGAGYHYYGMDVW

14
CAKQMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

15
CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGPARADLVGDAFDVW

16
CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW

17
CAKHMSMQDYLVIGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

TABLE 8

GLP1R Variants CDRH3 Sequences

SEQ ID

Variant
NO.
Sequence

GLP1R-1
18
CARANQHFVDLYGWHGVPKGYHYYGMDVW

GLP1R-2
19
CARDMYYDFETVVEGIQWYEALKAGKLGEVVPADDAFDIW

GLP1R-3
20
CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

GLP1R-8
21
CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW

GLP1R-10
22
CARANQHFFVPGSLKVWLKGVAPESSSEYDSSEDSGGAFDIW

GLP1R-25
23
CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW

GLP1R-26
24
CAKHMSMQEGVLQGQIPSTIDWEGLLHLIRADLVGDAFDVW

GLP1R-30
25
CARDMYYDFLKIGDNLAARDFINWLIQTKITDGTDTEVVPADDAFDIW

GLP1R-50
26
CARDGRGSLPRPKGGPKFVPGKHETYGHKTGYRLRPGYHYYGMDVW

GLP1R-56
27
CARANQHFFSGAEGEGQAAKEFIAWLVKGITGYHYYGMDVW

GLP1R-58
28
CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW

GLP1R-60
29
CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW

GLP1R-70
30
CARDGRGSLPRPKGGPPSSGRDFINWLIQTKITDGFRYDSSEDSGGAFDIW

GLP1R-71
31
CARDLRELECEEWTRHGGKKHHGKRQSNRAHQGKHETYGHKTGSLVPSRGPCVD

PRGVAGSFDVW

GLP1R-72
32
CARDMYYDFHPEGTFTSDVSSYLEGQAAKEFIAWLVKGSLIYEVVPADDAFDIW

GLP1R-80
33
CARANQHFGPVAGGATPSEEPGSQLTRAELGWDAPPGQESLADELLQLGTEHGYH

YYGMDVW

GLP1R-83
34
CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

GLP1R-93
35
CARANQHFLSHAGAARDFINWLIQTKITGLGSGYHYYGMDVW

GLP1R-98
36
CARDGRGSLPRPKGGPHSGRLGSGYKSYDSSEDSGGAFDIW

GLP1R-238
37
CARANQHFSQAGRAARVPGPSSSLGPRGYHYYGMDVW

GLP1R-239
38
CAKHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVW

GLP1R-240
39
CARDMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDAFDIW

GLP1R-241
40
CAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFD

VW

GLP1R-242
41
CAKHMSMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFDVW

GLP1R-243
42
CARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVW

GLP1R-244
43
CARANQHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVW

*bold corresponds to GLP1 or GLP2 motif

TABLE 9

Variable Heavy Chain Sequences

SEQ ID

Variant
NO.
Variable Heavy Chain Sequence

GLP1R-
44
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGSFSSHAISW

238

VRQAPGQGLEWMGGIIPIFGAPNYAQKFQGRVTITADESTSTAYMELSSLRSEDTA

VYYCARANQHFSQAGRAARVPGPSSSLGPRGYHYYGMDVWGQGTLVTVSSASAS

TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV

AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK

PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREP

QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-
45
MEWSWVFLFFLSVTTGVHSQVQLVESGGGVVQPGRSLRLSCAASGFDFSNYGMH

239

WVRQAPGKGLEWVADISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAE

DTAVYYCAKHMSMQSQGLDNLAARDFINWLIQTKITDGFELSRADLVGDAFDVW

GQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW

YVDGVEVHNAKTKPREEQFNSITRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP

IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE

NNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

LSPG

GLP1R-
46
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFNNYGIS

240

WVRQAPGQGLEWMGGIIPVFGTANYAQKFQGRVTITADESTSTAYMELSSLRSED

TAVYYCARDMYYDFFGLGTFTSDVSSYLEGQAAKEFIAWLVKGVSPEVVPADDA

FDIWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD

KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNK

GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPG

GLP1R-
47
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAIS

241

WVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDT

AVYYCAKHMSMQGSVAGGTFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVG

DAFDVWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT

VSWNSGALTSGVHIFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK

VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKV

SNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPG

GLP1R-
48
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYEISW

242

VRQAPGQGLEWMGGIIPILGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTA

VYYCAKHMSMQADTGTFTSDVSSYLEGQAAKEFIAWLVKGEFSSRADLVGDAFD

VWGQGTLVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN

SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV

ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP

APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG

GLP1R-
49
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGIN

243

WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDT

AVYYCARANQHFFGKGDNLAARDFINWLIQTKITDGSNPGYHYYGMDVWGQGT

LVTVSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG

VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV

ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG

VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI

SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-
50
MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW

244

VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTA

VYYCARANQHFAATGAGEGQAAKEFIAWLVKGRVEIGYHYYGMDVWGQGTLVT

VSSASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP

PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT

KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-
51
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVAVISYD

59-2

AGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDMYYDFETVV

EGIQWYEALKAGKLGEVVPADDAFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTS

ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSN

FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT

VVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ

VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

GLP1R-
52
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQGLEWMGGITIF

59-241

GTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQGSVAGG

TFTSDVSSYLEGQAAKEFIAWLVKGGPSFIRADLVGDAFDVWGQGTLVTVSSASA

STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP

VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT

KPREEQFNSITRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE

PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-
53
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGINWVRQAPGQGLEWMGGIIPIF

59-243

GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARANQHFFGKGDNL

AARDFINWLIQTKITDGSNPGYHYYGMDVWGQGTLVTVSSASASTKGPSVFPLAP

CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR

VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-3
54
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVSFISYDE

SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSSASTKGPSVFPLAPC

SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHIFPAVLQSSGLYSLSSVVT

VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV

VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

GLP1R-
55
MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGW

43-8

FRQAPGKEREGVAAINNFGTTKYADSAKGRFTISADNAKNTVYLQMNSLKPEDTA

VYYCAAVRWGPHNDDRYDWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE

PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

GLP1R-
56
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYDMEWVRQAPGKGLEWVAVISYE

10

GSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARANQHFFVPGSL

KVWLKGVAPESSSEYDSSEDSGGAFDIWGQGTLVTVSS

GLP1R-
57
QVQLVQSGAEVKKPGSSVKVSCKASGGTRSNYAINWVRQAPGQGLEWMGGIIPIL

26

GTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKHMSMQEGVLQG

QIPSTIDWEGLLHLIRADLVGDAFDVWGQGTLVTVSS

GLP1R-
58
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYAMEWVRQAPGKGLEWVAVISY

221-065

DRSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGDGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
59
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYPMEWVRQAPGKGLEWVAVISYD

221-075

ETNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAAKEFIAWLVKGIVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
60
QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYGVHWVRQAPGKGLEWVAFISYD

221-017

ESNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEYQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
61
QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYAMEWVRQAPGKGLEWVAVISHD

221-033

RSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAAKDFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
62
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYPMEWVRQAPGKGLEWVAVISYD

221-076

ETNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAAKEFIAWLVKGIVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
63
QVQLVESGGGVVQPGRSLRLSCAASGFIFNNYGMEWVRQAPGKGLEWVAFISYG

221-092

GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEGQAVKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
64
QVQLVESGGGVVQPGRSLRLSCAASGFPFSNYGMEWVRQAPGKGLEWVAVISHD

221-034

RSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAVKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
65
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYAMEWVRQAPGKGLEWVAVISY

221-066

DRSNEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEGQAIKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
66
QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGMEWVRQAPGKGLEWVAVISSD

221-084

ENNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEMQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
67
QVQLVESGGGVVQPGRSLRLSCAASGFIFSNYGMEWVRQAPGKGLEWVAVISDE

221-009

GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGAGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
68
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYPMEWVRQAPGKGLEWVAVISYD

222-052

ESNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GGGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
69
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYAMEWVRQAPGKGLEWVAVISDE

222-016

GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEYQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
70
QVQLVESGGGVVQPGRSLRLSCAASGFSFSDYGMEWVRQAPGKGLEWVAFISYD

222-023

ANNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEWQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
71
QVQLVESGGGVVQPGRSLRLSCAASGFAFSNYGMEWVRQAPGKGLEWVSFISYD

222-014

ESNKYYADSVKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAKHMSMQEGAV

TGEWQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
72
QVQLVESGGGVVQPGRSLRLSCAASGFSFSDYGIHWVRQAPGKGLEWVALISYEG

222-090

SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEKQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
73
QVQLVESGGGVVQPGRSLRLSCAASGFTFRDYGMEWVRQAPGKGLEWVAFIRYD

222-073

EINKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GEGQAAKEFIAWLVGGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
74
QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMEWVRQAPGKGLEWVAVISDE

222-012

GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGVGQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
75
QVQLVESGGGVVQPGRSLRLSCAASGFTFSAYSMEWVRQAPGKGLEWVALISYD

222-082

ATNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEFQAAKEFIAWLVKGRVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
76
QVQLVESGGGVVQPGRSLRLSCAASGFTFDNYALHWVRQAPGKGLEWVALISYD

222-081

AGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAV

TGEGQAAKEFIAWLVKGFVRADLVGDAFDVWGQGTLVTVSS

GLP1R-
77
QVQLVESGGGVVQPGRSLRLSCAASGFPFSSYAMEWVRQAPGKGLEWVAVISYD

222-056

RSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHMSMQEGAVT

GYGQAAKEFIAWLVKGFVRADLVGDAFDVWGQGTLVTVSS

TABLE 10

Variable Light Chain Sequences

SEQ ID

Variant
NO.
Variable Light Chain Sequence

GLP1R-
78
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIANNYVSW

238

YQQLPGTAPKLLIYANNRRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGAW

DVRLDVGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLS

GLP1R-
79
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSTSNIEKNYVSW

239

YQQLPGTAPKWYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTW

ENRLSAVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG

STVEKTVAPTECS

GLP1R-
80
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSSIGNNYVSW

240

YQQLPGTAPKLLIYANNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATW

SSSPRGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG

STVEKTVAPTECS

GLP1R-
81
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGISSNIGNNYVSW

241

YQQLPGTAPKWYDDDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTW

DNILSAAVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT

VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGS

TVEKTVAPTECS

GLP1R-
82
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIENNDVSW

242

YQQLPGTAPKWYGNDQRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTW

DNTLSAGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG

STVEKTVAPTECS

GLP1R-
83
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSRSNIGKNYVSW

243

YQQLPGTAPKWYENNERPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCSSYT

TSNTQVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV

AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST

VEKTVAPTECS

GLP1R-
84
MSVPTQVLGLLLLWLTDARCQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNVVSW

244

YQQLPGTAPKWYDNDKRRSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGSW

DTSLSVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG

STVEKTVAPTECS

GLP1R-
85
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSQSVLTQPPS

59-243

VSAAPGQKVTISCSGSRSNIGKNYVSWYQQLPGTAPKWYENNERPSGIPDRFSGS

KSGTSATLGITGLQTGDEADYYCSSYTTSNTQVFGGGTKLTVLGQPKAAPSVTLFP

PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA

SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

GLP1R-
86
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSQSVLTQPPS

59-241

VSAAPGQKVTISCSGISSNIGNNYVSWYQQLPGTAPKLLIYDDDQRPSGIPDRFSGS

KSGTSATLGITGLQTGDEADYYCGTWDNILSAAVFGGGTKLTVLGQPKAAPSVTL

FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA

ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

GLP1R-
87
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSQSALTQPAS

59-2

VSGSPGQSITISCTGTSNDIGTYNYVSWYQQHPGKAPKLMIYDVSGRPSGVSNRFS

GSKSGNTASLTISGLQAEDEADYYCSSYTTSSTEVFGGGTKLTVLGQPKAAPSVTL

FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA

ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

GLP1R-
88
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSQSALTQPAS

59-2A

VSGSPGQSITISCTGTSNDIGTYNYVSWYQQHPGKAPKLMIYDVSGRPSGVSNRFS

GSKSGNTASLTISGLQAEDEADYYCSSYTTSSTEVFGGGTKLTVLGQPKAAPSVTL

FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA

ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

GLP1R-3
89
QSVLTQPPSVSAAPGQKVTISCSGSSSNIADNYVSWYQQLPGTAPKLLIYDNNKRPS

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNYLGAGVFGGGTKLTVLGQ

PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTP

SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

GLP1R-
90
EIVMTQSPATLSVSPGERATLSCRASHSVSSDLAWYQQKPGQAPRLLIYSASSRAT

10

GTARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPASTFGGGTKVEIK

GLP1R-
91
EIVMTQSPATLSVSPGERATLSCSASQSVSTKLAWYQQKPGQAPRLLIYGASTRAK

26

GTARFSGSGSGTEFTLTISLQSEDFAVYYCQHYHNWPLTFGGGTKVEIK

GLP1R-
92
QSVLTQPPSVSAAPGQKVTISCSGTTSNIANNFVSWYQQLPGTAPKLLIYDHNKRPS

221-065

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSAGAFGGGTKLTVL

GLP1R-
93
QSVLTQPPSVSAAPGQKVTISCSGSGSNIGNNDVSWYQQLPGTAPKLLIYDNDKRP

221-075

AGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSNYVFGGGTKLTVL

GLP1R-
94
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNTYVSWYQQLPGTAPKLLIYDDYKRPS

221-017

GTDRFSGSKSGTSATLGITGLQTGDEADYYCATWDATLNTGVFGGGTKLTVL

GLP1R-
95
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNEYVSWYQQLPGTAPKLLIYDNNKRV

221-033

SGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDTSLNVGVFGGGTKLTVL

GLP1R-
96
QSVLTQPPSVSAAPGQKVTISCSGTSSNIGNNDVSWYQQLPGTAPKLLIYENNKRH

221-076

SGIPDRFSGSKSGTSATLGITGLQTGDEADYYCLTWDHSLTAYVFGGGTKLTVL

GLP1R-
97
QSVLTQPPSVSAAPGQKVTISCSGTTSNIANNFVSWYQQLPGTAPKLLIYDNNKRPP

221-092

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSVGMFGGGTKLTVL

GLP1R-
98
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNPVSWYQQLPGTAPKLLIYENDNRPS

221-034

GTDRFSGSKSGTSATLGITGLQTGDEADYYCATWDRGLSTGVFGGGTKLTVL

GLP1R-
99
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYLSWYQQLPGTAPKLLIYENNKRPS

221-066

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGIWDRSLSAWVFGGGTKLTVL

GLP1R-
100
QSVLTQPPSVSAAPGQKVTISCSGSSSNIADNYVSWYQQLPGTAPKLLIYENNRRPS

221-084

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDVSLSVGMFGGGTKLTVL

GLP1R-
101
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNQYVSWYQQLPGTAPKLLIYDDHKRPS

221-009

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSVGEFGGGTKLTVL

GLP1R-
102
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGKRSVSWYQQLPGTAPKLLIYDNNKRAS

222-052

GTDRFSGSKSGTSATLGITGLQTGDEADYYCVTWDRSLSAGVFGGGTKLTVL

GLP1R-
103
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNDVSWYQQLPGTAPKLLIYDFNKRPS

222-016

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSVGMFGGGTKLTVL

GLP1R-
104
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNDVSWYQQLPGTAPKLLIYENTKRPS

222-023

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDAGLSTGVFGGGTKLTVL

GLP1R-
105
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNHDVSWYQQLPGTAPKLLIYDNNKRH

222-014

SGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSAGVFGGGTKLTVL

GLP1R-
106
QSVLTQPPSVSAAPGQKVTISCSGSSSNIADNYVSWYQQLPGTAPKLLIYDNNKRA

222-090

SGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDNRLSAGVFGGGTKLTVL

GLP1R-
107
QSVLTQPPSVSAAPGQKVTISCSGSGSNIGNNDVSWYQQLPGTAPKLLIYDNNKRA

222-073

SGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDRGPNTGVFGGGTKLTVL

GLP1R-
108
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNDVSWYQQLPGTAPKLLIYDDDKRPS

222-012

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSVGEFGGGTKLTVL

GLP1R-
109
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGSKYVSWYQQLPGTAPKLLIYDNNKRPS

222-082

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDISPSAWVFGGGTKLTVL

GLP1R-
110
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGSDYVSWYQQLPGTAPKLLIYDNNKRSS

222-081

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDESLRSWVFGGGTKLTVL

GLP1R-
111
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGSNYISWYQQLPGTAPKLLIYDNDKRPA

222-056

GTDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDTSLSVGEFGGGTKLTVL

TABLE 11

GLP1R Sequences

GLP1R
SEQ ID

Variant
NO
Sequence

GLP1R-40-
112
EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREFLAAITSG

01

GATTYDDNRKSRFTISADNSKNTAYLQMNSLKPEDTAVYYCWAALDGYGGRW

GQGTLVTVSS

GLP1R-40-
113
EVQLVESGGGLVQPGGSLRLSCAASGRTFRINRMGWFRQAPGKEREWVSTICSR

02

GDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYSGSWG

QGTLVTVSS

GLP1R-40-
114
EVQLVESGGGLVQPGGSLRLSCAASGRDFRVKNMGWFRQAPGKEREFVARITW

03

NGGSAYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARILSRNWG

QGTLVTVSS

GLP1R-40-
115
EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYTMGWFRQAPGKEREFVAAISSGG

04

RTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYEGSWGQ

GTLVTVSS

GLP1R-40-
116
EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYAMGWFRQAPGKEREFVAAISSGG

05

RTRYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGIWGQ

GTLVTVSS

GLP1R-40-
117
EVQLVESGGGLVQPGGSLRLSCAASGHTSDTYIMGWFRQAPGKEREFVSLINWSS

06

GKTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKGDYRGGYYYP

QTSQWGQGTLVTVSS

GLP1R-40-
118
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKEREFVATIPSGG

07

STYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGSWGQ

GTLVTVSS

GLP1R-40-
119
EVQLVESGGGLVQPGGSLRLSCAASGFTFGEFTMGWFRQAPGKERERVATITSGG

08

STNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVDDYSGSWGQ

GTLVTVSS

GLP1R-40-
120
EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAW

09

GDGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNN

WGQGTLVTVSS

GLP1R-40-
121
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRS

10

GSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTAR

MVDWGQGTLVTVSS

GLP1R-40-
122
EVQLVESGGGLVQPGGSLRLSCAASGVTLDDYAMGWFRQAPGKEREFVAAINRS

11

GSITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALE

ETRGSYDWGQGTLVTVSS

GLP1R-40-
123
EVQLVESGGGLVQPGGSLRLSCAASGLTFGIYAMGWFRQAPGKEREFVATISRSG

12

ASTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYNDYDRGH

DWGQGTLVTVSS

GLP1R-40-
124
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSDGMGWFRQAPGKERELVAAINRS

13

GSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTARPGIFTTAPV

EDWGQGTLVTVSS

GLP1R-40-
125
EVQLVESGGGLVQPGGSLRLSCAASGFTCGNYTMGWFRQAPGKERESVASITSG

14

GRTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLDGYTGSWG

QGTLVTVSS

GLP1R-40-
126
EVQLVESGGGLVQPGGSLRLSCAASGFTFNYYPMGWFRQAPGKEREWVATISRG

15

GGTYYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGIWG

QGTLVTVSS

GLP1R-40-
127
EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMGWFRQAPGKEREFVGFITGSG

16

GTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARRYGNLYNT

NNYDWGQGTLVTVSS

GLP1R-40-
128
EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAMGWFRQAPGKEREFVAAISWR

17

GGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKY

TWGQGTLVTVSS

GLP1R-40-
129
EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISSKG

18

GSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSV

GDWRWGQGTLVTVSS

GLP1R-40-
130
EVQLVESGGGLVQPGGSLRLSCAASGSRFSGRFNILNMGWFRQAPGKEREFVAAI

19

SRSGDTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGS

NVEGRWGQGTLVTVSS

GLP1R-40-
131
EVQLVESGGGLVQPGGSLRLSCAASGGTSNSYRMGWFRQAPGKEREFVAVISWT

20

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVALDGYSGSW

GQGTLVTVSS

GLP1R-40-
132
EVQLVESGGGLVQPGGSLRLSCAASGFNIGTYTMGWFRQAPGKEREFVAAIGSN

21

GLANYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYSGTWG

QGTLVTVSS

GLP1R-40-
133
EVQLVESGGGLVQPGGSLRLSCAASGRTFSVYAMGWFRQAPGKEREFVAGIHSD

22

GSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYMGTWG

QGTLVTVSS

GLP1R-40-
134
EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVMGWFRQAPGKERELVAAVRWS

23

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWEG

SEPVQHEYDWGQGTLVTVSS

GLP1R-40-
135
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVAAIYCS

24

DGSTQYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAEALDGYWGQG

TLVTVSS

GLP1R-40-
136
EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYAMGWFRQAPGKEREMVAAMR

25

WSGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDD

YDGLPIKYDWGQGTLVTVSS

GLP1R-40-
137
EVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERECVTAIFSDG

26

GTYYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYNGYWG

QGTLVTVSS

GLP1R-40-
138
EVQLVESGGGLVQPGGSLRLSCAASGIHFAISTMGWFRQAPGKEREIVTAINWSG

27

ARTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAKFVNTDSTWS

RSEMYTWGQGTLVTVSS

GLP1R-40-
139
EVQLVESGGGLVQPGGSLRLSCAASGLTFTSYAMGWFRQAPGKEREGVAVIDSD

28

GTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLDGYSGSWG

QGTLVTVSS

GLP1R-40-
140
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSG

29

GSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRW

GQGTLVTVSS

GLP1R-40-
141
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSGVMGWFRQAPGKEREFVAAINRS

30

GSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTWG

QGTLVTVSS

GLP1R-40-
142
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKERELVAAISSGG

31

STSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAMDGYSGSWGQ

GTLVTVSS

GLP1R-40-
143
EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREYVAAISGS

32

GSITNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESYGWGN

RHFNWGQGTLVTVSS

GLP1R-40-
144
EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAIRWS

33

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDVTDYER

ADWGQGTLVTVSS

GLP1R-40-
145
EVQLVESGGGLVQPGGSLRLSCAASGFAFSGYAMGWFRQAPGKEREFVAAISWS

34

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAFVTTNSDYDLG

RDWGQGTLVTVSS

GLP1R-40-
146
EVQLVESGGGLVQPGGSLRLSCAASGIPASIRTMGWFRQAPGKEREGVSWISSSD

35

GSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCVAALDGYSGSWGQ

GTLVTVSS

GLP1R-40-
147
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSLPMGWFRQAPGKERELVAIRWSG

36

GSTVYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRW

DWGQGTLVTVSS

GLP1R-40-
148
EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSWISTT

37

DGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGIW

GQGTLVTVSS

GLP1R-40-
149
EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYAMGWFRQAPGKEREFVTAIDSES

38

RTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAALLDGYLGTWGQ

GTLVTVSS

GLP1R-40-
150
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMGWFRQAPGKEREFLGSILWSD

39

DSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANLKQGSYGYRF

NDWGQGTLVTVSS

GLP1R-40-
151
EVQLVESGGGLVQPGGSLRLSCAASGTIVNIHVMGWFRQAPGKERELVAAITSGG

40

STSYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRHFE

YDWGQGTLVTVSS

GLP1R-40-
152
EVQLVESGGGLVQPGGSLRLSCAASGRSLGTYHMGWFRQAPGKEREGVSWISSS

41

DGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVLDGYSGSW

GQGTLVTVSS

GLP1R-40-
153
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDTGMGWFRQAPGKEREFVAAIRWS

42

GKETWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMYYTL

EEYEYDWGQGTLVTVSS

GLP1R-40-
154
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMGWFRQAPGKERECVAAISSSD

43

GRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNWG

QGTLVTVSS

GLP1R-40-
155
EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKEREFIATIFSGG

44

DTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWD

WGQGTLVTVSS

GLP1R-40-
156
EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKEREIVASITSGG

45

RKNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQ

GTLVTVSS

GLP1R-40-
157
EVQLVESGGGLVQPGGSLRLSCAASGHSFGNFPMGWFRQAPGKEREVIAAIDWS

46

GGSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGW

GQGTLVTVSS

GLP1R-40-
158
EVQLVESGGGLVQPGGSLRLSCAASGSSFRFRAMGWFRQAPGKEREFVAAINRG

47

GKISHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYIRPDTYLSRD

YRKYDWGQGTLVTVSS

GLP1R-40-
159
EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDS

48

DGRTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSW

GQGTLVTVSS

GLP1R-40-
160
EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVAGISWS

49

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLG

NDWGQGTLVTVSS

GLP1R-40-
161
EVQLVESGGGLVQPGGSLRLSCAASGITFRRYDMGWFRQAPGKEREGVAYISSSD

50

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDDYSGGWG

QGTLVTVSS

GLP1R-40-
162
EVQLVESGGGLVQPGGSLRLSCAASGLTLSNYAMGWFRQAPGKEREFVAAISRS

51

GSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEMSGISGWD

WGQGTLVTVSS

GLP1R-40-
163
EVQLVESGGGLVQPGGSLRLSCAASGYTTSINTMGWFRQAPGKEREVVAAISRTG

52

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRRFE

YDWGQGTLVTVSS

GLP1R-40-
164
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIKPDG

53

SITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELF

HDEYNWGQGTLVTVSS

GLP1R-40-
165
EVQLVESGGGLVQPGGSLRLSCAASGSIFSLNAMGWFRQAPGKERELVAGISSKG

54

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWG

QGTLVTVSS

GLP1R-40-
166
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYRMGWFRQAPGKEREAVAAIASM

55

GGLTYVADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGSW

GQGTLVTVSS

GLP1R-40-
167
EVQLVESGGGLVQPGGSLRLSCAASGFTFGAFTMGWFRQAPGKERERVAAITCS

56

GSTTYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCSAALDGYNGSWG

QGTLVTVSS

GLP1R-40-
168
EVQLVESGGGLVQPGGSLRLSCAASGIPSTIRAMGWFRQAPGKERESVGRIYWRD

57

DNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSWG

QGTLVTVSS

GLP1R-40-
169
EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREVVAGIAW

58

GDGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNN

YYYPISRDEYDWGQGTLVTVSS

GLP1R-43-
170
EVQLVESGGGLVQAGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREVVASISWS

1

GKSTYVADSVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAQRRWSQDW

GQGTQVTVSS

GLP1R-43-
171
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

2

GGSTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGERD

YVVGQGTQVTVSS

GLP1R-43-
172
EVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMGWFRQAPGKEREFVATITWS

3

GSSTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYREYGY

WGQGTQVTVSS

GLP1R-43-
173
EVQLVESGGGLVQAGGSLRLSCAASGSIFHINPMGWFRQAPGKERENAAINIFGT

4

TNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVDGGPLWDDGY

DWGQGTQVTVSS

GLP1R-43-
174
EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVASINIFG

5

TTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVGWGPHNDDRY

DWGQGTQVTVSS

GLP1R-43-
175
EVQLVESGGGLVQAGGSLRLSCAASGTTFSIYAMEWFRQAPGKERELVATISRSG

6

GTTYVADSVGGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASWYYRDDY

WGQGTQVTVSS

GLP1R-43-
176
EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNF

7

GTTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAVRWGPHNDDR

YDWGQGTQVTVSS

GLP1R-43-
177
EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNF

8

GTTKYADSAKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPHNDDR

YDWGQGTQVTVSS

GLP1R-43-
178
EVQLVESGGGLVQAGGSLRLSCAASGFILYGYAMGWFRQAPGKEREGVSSISPSD

9

ASTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLNTYSDSWG

QGTQVTVSS

GLP1R-43-
179
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREGVTAISTS

10

DGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARDGYSGSW

GQGTQVTVSS

GLP1R-43-
180
EVQLVESGGGLVQAGGSLRLSCAASGYTITNSYRMGWFRQAPGKEREFVAGITM

11

SGFNTRYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRGLAGPA

WGQGTQVTVSS

GLP1R-43-
181
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDNAMGWFRQAPGKEREFVSGISTS

12

GSTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAGGYDYW

GQGTQVTVSS

GLP1R-43-
182
EVQLVESGGGLVQAGGSLRLSCAASGRTFSYYHMGWFRQAPGKEREGVSWISSY

13

YSSTYYADSESGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSCSWG

QGTQVTVSS

GLP1R-43-
183
EVQLVESGGGLVQAGGSLRLSCAASGSPFRLYTMGWFRQAPGKEREVVAHIYSY

14

GSINYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALWGHSGDWG

QGTQVTVSS

GLP1R-43-
184
EVQLVESGGGLVQAGGSLRLSCAASGSTFDTYGMGWFRQAPGKEREFVASITWS

15

GSSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANRIHWSGFYY

WGQGTQVTVSS

GLP1R-43-
185
EVQLVESGGGLVQAGGSLRLSCAASGRTSSPYTMGWFRQAPGKEREFVSAISWS

16

GGSTVYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIRRAPYSRLE

TWGQGTQVTVSS

GLP1R-43-
186
EVQLVESGGGLVQAGGSLRLSCAASGSIFPINAMGWFRQAPGKEREGVAAITNFG

17

TTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDHY

DWGQGTQVTVSS

GLP1R-43-
187
EVQLVESGGGLVQAGGSLRLSCAASGRTFDTYAMGWFRQAPGKEREFVAAITW

18

GGGRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRDY

DYWGQGTQVTVSS

GLP1R-43-
188
EVQLVESGGGLVQAGGSLRLSCAASGRRFSAYGMGWFRQAPGKEREFVAAVSW

19

DGRNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTDDYGVDW

GQGTQVTVSS

GLP1R-43-
189
EVQLVESGGGLVQAGGSLRLSCAASGSTFDNYAMGWFRQAPGKEREFVSAISGD

20

GGTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRLYRNRD

YWGQGTQVTVSS

GLP1R-43-
190
EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVSWITSFD

21

ASTYYADSVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSGSWG

QGTQVTVSS

GLP1R-43-
191
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVSTISTG

22

GSSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTGRGRRD

YWGQGTQVTVSS

GLP1R-43-
192
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

23

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPVVPNTKD

YWGQGTQVTVSS

GLP1R-43-
193
EVQLVESGGGLVQAGGSLRLSCAASGNVFMIKDMGWFRQAPGKEREWVTAISW

24

NGGSTDYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIVTYSDYDL

GNDWGQGTQVTVSS

GLP1R-43-
194
EVQLVESGGGLVQAGGSLRLSCAASGFPFSIWPMGWFRQAPGKEREFIATIFSGG

25

DTDYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEEGVYRWD

WGQGTQVTVSS

GLP1R-43-
195
EVQLVESGGGLVQAGGSLRLSCAASGRGFSRYAMGWFRQAPGKEREFVAAIRW

26

SGKETWYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALGPVRRSRLE

WGQGTQVTVSS

GLP1R-43-
196
EVQLVESGGGLVQAGGSLRLSCAASGRTSDIYGMGWFRQAPGKEREFVARIYWS

27

SGNTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAYRFSDYSRP

AGYDWGQGTQVTVSS

GLP1R-43-
197
EVQLVESGGGLVQAGGSLRLSCAASGNDFSFNSMGWFRQAPGKEREFLASVSWG

28

FGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARAYGNPTWGQ

GTQVTVSS

GLP1R-43-
198
EVQLVESGGGLVQAGGSLRLSCAASGRTFTDYPMGWFRQAPGKERELESFVPIN

29

GTSTYYADSDSGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYSCSW

GQGTQVTVSS

GLP1R-43-
199
EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVATISRGG

30

STTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGKDYWG

QGTQVTVSS

GLP1R-43-
200
EVQLVESGGGLVQAGGSLRLSCAASGFIFQLYVMGWFRQAPGKEREGVTYINNI

31

DGSTYYAYSVRGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRDGYSGSW

GQGTQVTVSS

GLP1R-43-
201
EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYAMEWFRQAPGKERELVATISRSG

32

GRTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAANWYYRYDY

WGQGTQVTVSS

GLP1R-43-
202
EVQLVESGGGLVQAGGSLRLSCAASGFPFRINAMGWFRQAPGKERELVTAISSSG

33

SSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASGYYATYYGE

RDYVVGQGTQVTVSS

GLP1R-43-
203
EVQLVESGGGLVQAGGSLRLSCAASGFTLSSYTMGWFRQAPGKEREFVSAISRGG

34

GNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSYAEYDYW

GQGTQVTVSS

GLP1R-43-
204
EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAAINGG

35

GDSTNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAASASPYSGRN

YVVGQGTQVTVSS

GLP1R-43-
205
EVQLVESGGGLVQAGGSLRLSCAASGLLISTTVMGWFRQAPGKEREGDGYISITD

36

GSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAALDGYSGSWG

QGTQVTVSS

GLP1R-43-
206
EVQLVESGGGLVQAGGSLRLSCAASGRTLENYRMGWFRQAPGKEREFVAAVSW

37

SSGNAVYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAANWKMLLG

VENDWGQGTQVTVSS

GLP1R-43-
207
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

38

GGSTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTVYGERD

YWGQGTQVTVSS

GLP1R-43-
208
EVQLVESGGGLVQAGGSLRLSCAASGSILSISPMGWFRQAPGKERELVAINFSWG

39

TTDYADSvKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAYEQGVYRWD

WGQGTQVTVSS

GLP1R-43-
209
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

40

GGSTYVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAERYRYSGYY

ARDSWGQGTQVTVSS

GLP1R-43-
210
EVQLVESGGGLVQAGGSLRLSCAASGFTLSDYAMGWFRQAPGKEREFVSAISRD

41

GTTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPTSQYATD

YWGQGTQVTVSS

GLP1R-43-
211
EVQLVESGGGLVQAGGSLRLSCAASGRDLDYYVMGWFRQAPGKERELVAIKFS

42

GGTTDVADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCADIAYEEGVYR

WDWGQGTQVTVSS

GLP1R-43-
212
EVQLVESGGGLVQAGGSLRLSCAASGSIFTFNAMGWFRQAPGKEREFVAGITRSA

43

VSTSYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAFRGIMRPDWG

QGTQVTVSS

GLP1R-43-
213
EVQLVESGGGLVQAGGSLRLSCAASGRTFDSYAMGWFRQAPGKEREFVAAITSS

44

GGNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPARYGARD

YVVGQGTQVTVSS

GLP1R-43-
214
EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIG

45

GATNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDGRQVWGQ

GTQVTVSS

GLP1R-43-
215
EVQLVESGGGLVQAGGSLRLSCAASGGIFRKLAMGWFRQAPGKERELVAAIRW

46

SGGITWYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATLAKGGGR

WGQGTQVTVSS

GLP1R-43-
216
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

47

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSDRD

YWGQGTQVTVSS

GLP1R-43-
217
EVQLVESGGGLVQAGGSLRLSCAASGRTFRIYAMGWFRQAPGKERELVSSISWN

48

SGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAAYSYTQGTT

YESWGQGTQVTVSS

GLP1R-43-
218
EVQLVESGGGLVQAGGSLRLSCAASGRTFTSYRMGWFRQAPGKEREWMGTIDY

49

SGRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAMDGYSGSW

GQGTQVTVSS

GLP1R-43-
219
EVQLVESGGGLVQAGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAINWN

50

GDTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRYSDYDY

WGQGTQVTVSS

GLP1R-43-
220
EVQLVESGGGLVQAGGSLRLSCAASGRFFSTRVMGWFRQAPGKERELVAIKFSG

51

GTTDYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAIAHEEGVYRW

DWGQGTQVTVSS

GLP1R-43-
221
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

52

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGTRD

YWGQGTQVTVSS

GLP1R-43-
222
EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKEREGVSYISMS

53

DGRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAELDGYSGSW

GQGTQVTVSS

GLP1R-43-
223
EVQLVESGGGLVQAGGSLRLSCAASGLSFSGYTMGWFRQAPGKEREVVAAISRT

54

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCALIQRRAPYSRL

ETWGQGTQVTVSS

GLP1R-43-
224
EVQLVESGGGLVQAGGSLRLSCAASGSTLSIYGMGWFRQAPGKEREGVAAISWS

55

DGSTSYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVADIGLASDF

DYWGQGTQVTVSS

GLP1R-43-
225
EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYAMGWFRQAPGKEREFVATITRSS

56

GNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPFKPYSYDY

WGQGTQVTVSS

GLP1R-43-
226
EVQLVESGGGLVQAGGSLRLSCAASGSTFSIYTMGWFRQAPGKEREFVAAISGSS

57

DSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVPKTRYTRDY

WGQGTQVTVSS

GLP1R-43-
227
EVQLVESGGGLVQAGGSLRLSCAASGNTFSSYAMGWFRQAPGKEREFVAIISRSG

58

GRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAAPYNETNSWG

QGTQVTVSS

GLP1R-43-
228
EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVASISRSG

59

GRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARYNERNSWG

QGTQVTVSS

GLP1R-43-
229
EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAI

60

YWSNGKTPYADSVKRRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYS

GAWGQGTQVTVSS

GLP1R-43-
230
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

61

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPRAPSERDY

WGQGTQVTVSS

GLP1R-43-
231
EVQLVESGGGLVQAGGSLRLSCAASGRTFNNNDMGWFRQAPGKEREFVAVIKL

62

GGATTYDDYSEGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATWDARHVWG

QGTQVTVSS

GLP1R-43-
232
EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSS

63

DGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGCSGSW

GQGTQVTVSS

GLP1R-43-
233
EVQLVESGGGLVQAGGSLRLSCAASGSTFSTYAMGWFRQAPGKEREFVAAINRS

64

GASTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALLGGRGGC

GKGYWGQGTQVTVSS

GLP1R-43-
234
EVQLVESGGGLVQAGGSLRLSCAASGSILDTYAMGWFRQAPGKERELVSGINTS

65

GDTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLAGYEYWG

QGTQVTVSS

GLP1R-43-
235
EVQLVESGGGLVQAGGSLRLSCAASGSTLSINAMGWFRQAPGKEREFVAHMSHD

66

GTTNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCARLPNYRWGQGT

QVTVSS

GLP1R-43-
236
EVQLVESGGGLVQAGGSLRLSCAASGSIFRLNAMGWFRQAPGKEREGVAAINNF

67

DTTKYADSSKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRSDDR

WGQGTQVTVSS

GLP1R-43-
237
EVQLVESGGGLVQAGGSLRLSCAASGLTNPPFDNFPMGWFRQAPGKEREFVAVIS

68

WTGGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCPAVYPRYYG

DDDRPPVDWGQGTQVTVSS

GLP1R-43-
238
EVQLVESGGGLVQAGGSLRLSCAASGPTFSKAVMGWFRQAPGKEREFVAAMNW

69

SGRSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATPAGRGGY

WGQGTQVTVSS

GLP1R-43-
239
EVQLVESGGGLVQAGGSLRLSCAASGSIFSDYAMGWFRQAPGKEREFVATINWG

70

GGRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYARD

YWGQGTQVTVSS

GLP1R-43-
240
EVQLVESGGGLVQAGGSLRLSCAASGFILSDYAMGWFRQAPGKEREFVAAISSSE

71

ASTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRFWAGYDSW

GQGTQVTVSS

GLP1R-43-
241
EVQLVESGGGLVQAGGSLRLSCAASGYTDYKYDMGWFRQAPGKEREFVAAISW

72

GGGLTVYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVATVTDYT

GTYSDGWGQGTQVTVSS

GLP1R-43-
242
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVATINW

73

GGGNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYAY

DYWGQGTQVTVSS

GLP1R-43-
243
EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYYMGWFRQAPGKERELVAVILRG

74

GSTNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAARRYGNLYNT

NNYDWGQGTQVTVSS

GLP1R-43-
244
EVQLVESGGGLVQAGGSLRLSCAASGSILSSYVMGWFRQAPGKEREFVSAISRSG

75

TSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYDRDY

WGQGTQVTVSS

GLP1R-43-
245
EVQLVESGGGLVQAGGSLRLSCAASGFTLDNYAMGWFRQAPGKEREFVAAISWS

76

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYSYD

YWGQGTQVTVSS

GLP1R-43-
246
EVQLVESGGGLVQAGGSLRLSCAASGNTYSYKVMGWFRQAPGKEREFVGIIIRN

77

GDTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASPKYMTAYE

RSYDWGQGTQVTVSS

GLP1R-43-
247
EVQLVESGGGLVQAGGSLRLSCAASGSIFRNYAMGWFRQAPGKEREFVATITTSG

78

GNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPKTRYRRDY

WGQGTQVTVSS

GLP1R-43-
248
EVQLVESGGGLVQAGGSLRLSCAASGFTFGTTTMGWFRQAPGKEREVVAAITGS

79

GRSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAASAIGSGALRR

FEYDWGQGTQVTVSS

GLP1R-43-
249
EVQLVESGGGLVQAGGSLRLSCAASGGTFSAYAMGWFRQAPGKEREGVAAIRW

80

DGGYTRYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAATTPTTSYLP

RSERQYEWGQGTQVTVSS

GLP1R-43-
250
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

81

GGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVPSVYGERD

YWGQGTQVTVSS

GLP1R-43-
251
EVQLVESGGGLVQAGGSLRLSCAASGSFFSINAMGWFRQAPGKEREFVAGISQSG

82

GSTAYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAHRIVVGGTSVG

DWRWGQGTQVTVSS

GLP1R-43-
252
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREMVASITSR

83

KIPKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVWSGRDWGQGT

QVTVSS

GLP1R-43-
253
EVQLVESGGGLVQAGGSLRLSCAASGFTFRRYVMGWFRQAPGKEREFVAAISRD

84

GDRTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASTRLAGRWYR

DSEYKWGQGTQVTVSS

GLP1R-43-
254
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDNAMGWFRQAPGKEREFVATISRG

85

GSRTSYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAGPRSGRDYW

GQGTQVTVSS

GLP1R-43-
255
EVQLVESGGGLVQAGGSLRLSCAASGFTFRSYAMGWFRQAPGKEREFVATITRN

86

GDNTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCATVGTRYNYW

GQGTQVTVSS

GLP1R-43-
256
EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYVMGWFRQAPGKERELISGITWN

87

GDTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVVRLGGYDY

WGQGTQVTVSS

GLP1R-43-
257
EVQLVESGGGLVQAGGSLRLSCAASGGIISNYHMGWFRQAPGKEREFVATITRSG

88

GSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAMAGRGRWGQG

TQVTVSS

GLP1R-43-
258
EVQLVESGGGLVQAGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVSAIG

89

WSGASTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAYYTDYDE

ALEETRGSYDWGQGTQVTVSS

GLP1R-43-
259
EVQLVESGGGLVQAGGSLRLSCAASGSTFPIYAMGWFRQAPGKEREWVSGISSR

90

DDTTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCSAHRIVFRGTSV

GDWRWGQGTQVTVSS

GLP1R-43-
260
EVQLVESGGGLVQAGGSLRLSCAASGRAFSYYNMGWFRQAPGKEREGVSWISSS

91

DGSTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVLDGYSGSW

GQGTQVTVSS

GLP1R-43-
261
EVQLVESGGGLVQAGGSLRLSCAASGSTFSIDVMGWFRQAPGKERELVAATGRR

92

GGPTYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAARTSYSGTYD

YGVDWGQGTQVTVSS

GLP1R-43-
262
EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAINWS

93

GSITYYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAVGRSGRDYWG

QGTQVTVSS

GLP1R-43-
263
EVQLVESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREGVAAINNF

94

GTTKYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAVRWGPRNDDR

YDWGQGTQVTVSS

GLP1R-43-
264
EVQLVESGGGLVQAGGSLRLSCAASGGTLNNNPMAMGWFRQAPGKEREFVVAI

95

YWSNGKTQYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCAAALDGYS

GSWGQGTQVTVSS

GLP1R-43-
265
EVQLVESGGGLVQAGGSLRLSCAASGRTFNNDHMGWFRQAPGKEREFVAVIEIG

96

GATNYADSVKGRFTISADNAKNTVYLQMNSLKPEDTAVYYCASWDGRQVWGQ

GTQVTVSS

GLP1R-41-
266
EVQLVESGGGLVQPGGSLRLSCAASGRTFAMGWMGWFRQAPGKEREFVARVS

01

WDGRNAYYANSRFGRFTISADNSKNTAYLQMNSLKPEDTAVYYCPRYVSPARD

HGCWGQGTLVTVSS

GLP1R-41-
267
EVQLVESGGGLVQPGGSLRLSCAASGLTISTYIMGWFRQAPGKEREFVAVVNWN

02

GDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEAL

EETRGSYDWGQGTLVTVSS

GLP1R-41-
268
EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWFRQAPGKERELVAAINRG

03

GKITHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASLRNSGSNVE

GRWGQGTLVTVSS

GLP1R-41-
269
EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREFVAAISPS

04

AVTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYDYYSDYPLP

DANEYEWGQGTLVTVSS

GLP1R-41-
270
EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYIMGWFRQAPGKEREFVAVINRSG

05

STTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSNSSDYY

SQEGAYDWGQGTLVTVSS

GLP1R-41-
271
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYVMGWFRQAPGKEREGVSYISSSD

06

GRTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYNGSWG

QGTLVTVSS

GLP1R-41-
272
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRFGMGWFRQAPGKEREGVAAIGSD

07

GSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASGRDRYARDLSE

YEYVWGQGTLVTVSS

GLP1R-41-
273
EVQLVESGGGLVQPGGSLRLSCAASGFTFRFNAMGWFRQAPGKEREFVAAINWR

08

GSHPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATLGEPLVKY

TWGQGTLVTVSS

GLP1R-41-
274
EVQLVESGGGLVQPGGSLRLSCAASGGTFGVYHMGWFRQAPGKEREFLASVTW

09

GFGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATTTRSYDDT

YRNSWVYNWGQGTLVTVSS

GLP1R-41-
275
EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAIRWS

10

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSDYLP

MDWGQGTLVTVSS

GLP1R-41-
276
EVQLVESGGGLVQPGGSLRLSCAASGPTFTIYAMGWFRQAPGKEREFVGAISMSG

11

EDTIYADSEKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYTSNTNYY

NQEGAYDWGQGTLVTVSS

GLP1R-41-
277
EVQLVESGGGLVQPGGSLRLSCAASGPTFSNYYVGWFRQAPGKEREFVAAILCSG

12

GITCYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYIGTWGQ

GTLVTVSS

GLP1R-41-
278
EVQLVESGGGLVQPGGSLRLSCAASGGTFSSIGMGWFRQAPGKEREGVAAIGSD

13

GSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDRYARVLTE

YEYVWGQGTLVTVSS

GLP1R-41-
279
EVQLVESGGGLVQPGGSLRLSCAASGVTFNNYGMGWFRQAPGKERELVAAIRW

14

SGSATFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDGARGSW

GQGTLVTVSS

GLP1R-41-
280
EVQLVESGGGLVQPGGSLRLSCAASGRTFTMDGMGWFRQAPGKEREGVAAIGS

15

DGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSNIGGSRWR

YDWGQGTLVTVSS

GLP1R-41-
281
EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAAISPAA

16

LTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYLPSPYYSSYY

DSTKYEWGQGTLVTVSS

GLP1R-41-
282
EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKEREVVAAISWN

17

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASAIGSGALRR

FEYDWGQGTLVTVSS

GLP1R-41-
283
EVQLVESGGGLVQPGGSLRLSCAASGFTFGFYAMGWFRQAPGKERELVAAISWS

18

DASTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDY

YNVSEYDWGQGTLVTVSS

GLP1R-41-
284
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYPMGWFRQAPGKERECVSTIWSRG

19

DTYYADNVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSATWGQ

GTLVTVSS

GLP1R-41-
285
EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAMGWFRQAPGKERELVAAISWS

20

NDITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDYY

SVSEYDWGQGTLVTVSS

GLP1R-41-
286
EVQLVESGGGLVQPGGSLRLSCAASGGTFSTYTMGWFRQAPGKEREFVAGIYND

21

GTASYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYTGNDW

GQGTLVTVSS

GLP1R-41-
287
EVQLVESGGGLVQPGGSLRLSCAASGVTLDLYAMGWFRQAPGKEREWVARMY

22

LDGDYPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSG

SWGQGTLVTVSS

GLP1R-41-
288
EVQLVESGGGLVQPGGSLRLSCAASGRTISRYIMGWFRQAPGKERELVAAINRSG

23

KSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWYRDS

EYKWGQGTLVTVSS

GLP1R-41-
289
EVQLVESGGGLVQPGGSLRLSCAASGRTLSVYAMGWFRQAPGKEREFVAAVRW

24

SGGITWYVDSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFDGYSGSD

WGQGTLVTVSS

GLP1R-41-
290
EVQLVESGGGLVQPGGSLRLSCAASGSIFSITEMGWFRQAPGKERELVAAIAVGG

25

GITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHDVDDDESPY

YSGGYYRALYDWGQGTLVTVSS

GLP1R-41-
291
EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKERELVAAISPAA

26

LTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASMSLRPLDPAS

YSPDIQPYDWGQGTLVTVSS

GLP1R-41-
292
EVQLVESGGGLVQPGGSLRLSCAASGFTCGDYTMGWFRQAPGKERESVAAIDSD

27

GRTHYADSVISRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGDWGQ

TLVTVSS

GLP1R-41-
293
EVQLVESGGGLVQPGGSLRLSCAASGRTLSfYAMGWFRQAPGKEREFVAAINRG

28

GRISHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRRYGSPPHD

GSSYEWGQGTLVTVSS

GLP1R-41-
294
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKEREFVAGISWT

29

GGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNVGFEWGQG

TLVTVSS

GLP1R-41-
295
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMGWFRQAPGKEREGVAAIGSD

30

GSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATLRATITNFDEY

VWGQGTLVTVSS

GLP1R-41-
296
EVQLVESGGGLVQPGGSLRLSCAASGRTFNRYPMGWFRQAPGKEREFVAHMSH

31

DGTTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAPGTRYYGSN

QVNYNWGQGTLVTVSS

GLP1R-41-
297
EVQLVESGGGLVQPGGSLRLSCAASGSIFSFNAMGWFRQAPGKEREFVAGITRRG

32

LSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAKGIGVYGWG

QGTLVTVSS

GLP1R-41-
298
EVQLVESGGGLVQPGGSLRLSCAASGGSISSINAMGWFRQAPGKERELVAGIITSG

33

DSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGSAYVAGVRR

RNAYHWGQGTLVTVSS

GLP1R-41-
299
EVQLVESGGGLVQPGGSLRLSCAASGGTFSADVMGWFRQAPGKEREFVAAISTG

34

SITIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATYGYDSGLYFITDS

NDYEWGQGTLVTVSS

GLP1R-41-
300
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAMRW

35

RGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGTLYDDY

DGLPIKYDWGQGTLVTVSS

GLP1R-41-
301
EVQLVESGGGLVQPGGSLRLSCAASGDIFNINAMGWFRQAPGKEREPVAAISPAA

36

LTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAATPIERLGLDAYE

YDWGQGTLVTVSS

GLP1R-41-
302
EVQLVESGGGLVQPGGSLRLSCAASGRTFSTYNMGWFRQAPGKEREFVAAINWS

37

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPPDSSWYLD

SPEFFKWGQGTLVTVSS

GLP1R-41-
303
EVQLVESGGGLVQPGGSLRLSCAASGSISVFDAMGWFRQAPGKERELVAGISGSG

38

GDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASPKYSTHSIFD

ASPYNWGQGTLVTVSS

GLP1R-41-
304
EVQLVESGGGLVQPGGSLRLSCAASGFTSDDYAMGWFRQAPGKEREFVAALRW

39

SSSN1DYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGHG

DVSEYEYDWGQGTLVTVSS

GLP1R-41-
305
EVQLVESGGGLVQPGGSLRLSCAASGFTFSPNVMGWFRQAPGKEREFVAAITSSG

40

ETTWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSSLMS

EYDWGQGTLVTVSS

GLP1R-41-
306
EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAINW

41

SGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLLGV

ENDWGQGTLVTVSS

GLP1R-41-
307
EVQLVESGGGLVQPGGSLRLSCAASGDTFNCYAMGWFRQAPGKEREFVAVINW

42

SGDNTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEA

LEETRGRYDWGQGTLVTVSS

GLP1R-41-
308
EVQLVESGGGLVQPGGSLRLSCAASGSISTINVMGWFRQAPGKEREFVAAISPSA

43

VTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSEY

EYDWGQGTLVTVSS

GLP1R-41-
309
EVQLVESGGGLVQPGGSLRLSCAASGRTLSKYRMGWFRQAPGKEREFVAAIRWS

44

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIPHGIAGRITWG

QGTLVTVSS

GLP1R-41-
310
EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYAMGWFRQAPGKERELVAGIDQS

45

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADDYLGGDNW

YLGPYDWGQGTLVTVSS

GLP1R-41-
311
EVQLVESGGGLVQPGGSLRLSCAASGFTIDDYAMGWFRQAPGKEREFVAAVSGT

46

GTIAYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYIDYDEALE

ETRGSYDWGQGTLVTVSS

GLP1R-41-
312
EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYVMGWFRQAPGKERELVAGITSG

47

RDITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADGVLATTLNW

DWGQGTLVTVSS

GLP1R-41-
313
EVQLVESGGGLVQPGGSLRLSCAASGSGISFNAMGWFRQAPGKERELVAAISRSG

48

DTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLTTWADGPY

RWGQGTLVTVSS

GLP1R-41-
314
EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRG

49

GKISHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGNPPHD

GSSYEWGQGTLVTVSS

GLP1R-41-
315
EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGG

50

TTDYADSvkGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWGQ

GTLVTVSS

GLP1R-41-
316
EVQLVESGGGLVQPGGSLRLSCAASGGIFRFNAMGWFRQAPGKERELVAGISGSG

51

GDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWG

QGTLVTVSS

GLP1R-41-
317
EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYAMGWFRQAPGKEREFVAAINRG

52

GKISHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVRRYGSPPHD

GSSYEWGQGTLVTVSS

GLP1R-41-
318
EVQLVESGGGLVQPGGSLRLSCAASGSDFSLNAMGWFRQAPGKEREFVAAISWS

53

GGSTLYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASNESDAYNWG

QGTLVTVSS

GLP1R-41-
319
EVQLVESGGGLVQPGGSLRLSCAASGRTLVNYDMGWFRQAPGKEREFVAAIRW

54

SGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMLPPW

GQGTLVTVSS

GLP1R-41-
320
EVQLVESGGGLVQPGGSLRLSCAASGRTFEKDAMGWFRQAPGKEREMVAAIRW

55

SGGITCYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSLPDDYD

GLECEYDWGQGTLVTVSS

GLP1R-41-
321
EVQLVESGGGLVQPGGSLRLSCAASGSFFKINAMGWFRQAPGKEREFVAGITRSG

56

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAESLGRWWGQG

TLVTVSS

GLP1R-41-
322
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWS

57

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDWGQG

TLVTVSS

GLP1R-41-
323
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVAAIRWS

58

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASHDSDYGGT

NANLYDWGQGTLVTVSS

GLP1R-41-
324
EVQLVESGGGLVQPGGSLRLSCAASGRTDRSNVMGWFRQAPGKEREFVAAINRS

59

GSTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTAR

MVDWGQGTLVTVSS

GLP1R-41-
325
EVQLVESGGGLVQPGGSLRLSCAASGSFFSINVMGWFRQAPGKERELVAATGRR

60

GGPTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSV

GDWRWGQGTLVTVSS

GLP1R-41-
326
EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREGVAAIDS

61

DGRTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGNW

GQGTLVTVSS

GLP1R-41-
327
EVQLVESGGGLVQPGGSLRLSCAASGNIFSLNTMGWFRQAPGKEREFVAAINCSG

62

NHPYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDDDGRD

NVVGQGTLVTVSS

GLP1R-41-
328
EVQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAVSGSG

63

DDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVQAYSSSSDYY

SQEGAYDWGQGTLVTVSS

GLP1R-41-
329
EVQLVESGGGLVQPGGSLRLSCAASGFTFPAYVMGWFRQAPGKERELLAVITRD

64

GSTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRIWSSR

NPWGQGTLVTVSS

GLP1R-41-
330
EVQLVESGGGLVQPGGSLRLSCAASGFSFDDDYVMGWFRQAPGKERELVAVIG

65

WGGKETWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEDPSMGY

YTLEEYEYDWGQGTLVTVSS

GLP1R-41-
331
EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISMS

66

GDDTAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLRGRGDVS

EYEYDWGQGTLVTVSS

GLP1R-41-
332
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIDAMGWFRQAPGKEREFVGAITWG

67

GGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTDGDYDG

WGQGTLVTVSS

GLP1R-41-
333
EVQLVESGGGLVQPGGSLRLSCAASGNTFSINVMGWFRQAPGKEREFVAAINWN

68

GGSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLD

NDWGQGTLVTVSS

GLP1R-41-
334
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTHWMGWFRQAPGKEREVVAVIYTS

69

DGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANEYGLGSSIY

AYKWGQGTLVTVSS

GLP1R-41-
335
EVQLVESGGGLVQPGGSLRLSCAASGRTFSISAMGWFRQAPGKEREFVAAISRSG

70

GTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNEY

DWGQGTLVTVSS

GLP1R-41-
336
EVQLVESGGGLVQPGGSLRLSCAASGSTFRINAMGWFRQAPGKERELVAAISPAA

71

LTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAEPYGSGSLYDD

YDGLPIKYDWGQGTLVTVSS

GLP1R-41-
337
EVQLVESGGGLVQPGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVAAISWS

72

NDITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALSEVWRGSE

NLREGYDWGQGTLVTVSS

GLP1R-41-
338
EVQLVESGGGLVQPGGSLRLSCAASGLPVDYYAMGWFRQAPGKERELVAAISGS

73

GDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQTEDSASIFG

Y

GMDWGQGTLVTVSS

GLP1R-41-
339
EVQLVESGGGLVQPGGSLRLSCAASGRTLSTVNMGWFRQAPGKEREFVGAISRS

74

GETTWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVDCPDYYSDY

ECPLEWGQGTLVTVSS

GLP1R-41-
340
EVQLVESGGGLVQPGGSLRLSCAASGFSFDDYAMGWFRQAPGKERELVAAVRW

75

SGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGDTGGAAY

GWGQGTLVTVSS

GLP1R-41-
341
EVQLVESGGGLVQPGGSLRLSCAASGSTLSINAMGWFRQAPGKEREGVSWISSSD

76

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGRWG

QGTLVTVSS

GLP1R-41-
342
EVQLVESGGGLVQPGGSLRLSCAASGSSVSIDAMGWFRQAPGKEREFVAGISRSG

77

DTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASYNVYYNNYY

YPISRDEYDWGQGTLVTVSS

GLP1R-41-
343
EVQLVESGGGLVQPGGSLRLSCAASGSIFRVNVMGWFRQAPGKERELVAVTWSG

78

GSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRW

DWGQGTLVTVSS

GLP1R-41-
344
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAVVNWS

79

GRRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASSRMGVDDP

ETYGWGQGTLVTVSS

GLP1R-41-
345
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDAAMGWFRQAPGKEREFVAAVRW

80

RGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDY

DGLPIKYDWGQGTLVTVSS

GLP1R-41-
346
EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWFRQAPGKERELVASISRFG

81

RTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANGIESWGQGTLV

TVSS

GLP1R-41-
347
EVQLVESGGGLVQPGGSLRLSCAASGFTWGDYTMGWFRQAPGKEREFVASITSG

82

GRMWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSW

GQGTLVTVSS

GLP1R-41-
348
EVQLVESGGGLVQPGGSLRLSCAASGFRFSSYGMGWFRQAPGKEREGVAAIGSD

83

GSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQG

TLVTVSS

GLP1R-41-
349
EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYNMGWFRQAPGKEREFVAAISW

84

NGVTIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDW

GQGTLVTVSS

GLP1R-41-
350
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYSMGWFRQAPGKEREFVAAISSGG

85

LKAYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDDYSGSWGQ

GTLVTVSS

GLP1R-41-
351
EVQLVESGGGLVQPGGSLRLSCAASGYTFRAYVMGWFRQAPGKERELLAVITRD

86

GSTHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVNGRWRSWSSR

NPWGQGTLVTVSS

GLP1R-41-
352
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKEREFVAAISRGS

87

NSTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYTDYDLWG

QGTLVTVSS

GLP1R-41-
353
EVQLVESGGGLVQPGGSLRLSCAASGRTISSYAMGWFRQAPGKERELVAAISKSS

88

ISTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLEWG

QGTLVTVSS

GLP1R-41-
354
EVQLVESGGGLVQPGGSLRLSCAASGPTFDTYVMGWFRQAPGKEREFVAAISWT

89

GDSSSDGDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAIFDV

TDYERADWGQGTLVTVSS

GLP1R-41-
355
EVQLVESGGGLVQPGGSLRLSCAASGFTLGNYAMGWFRQAPGKERELVSAITWS

90

DGSSYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASTRFAGRWGQ

GTLVTVSS

GLP1R-41-
356
EVQLVESGGGLVQPGGSLRLSCAASGNIDRLYAMGWFRQAPGKEREPVAAISPA

91

AVTAGMTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYGSGSY

YYTDDELDWGQGTLVTVSS

GLP1R-41-
357
EVQLVESGGGLVQPGGSLRLSCAASGRTFGRRAMGWFRQAPGKERELVAAIRWS

92

GKETWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRTYGH

SRARYEWGQGTLVTVSS

GLP1R-41-
358
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIGAMGWFRQAPGKEREYVGSITWR

93

GGNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGVTGGAAYG

WGQGTLVTVSS

GLP1R-41-
359
EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYWMGWFRQAPGKEREVVAVIYTS

94

DGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATIDGSWREWG

GTLVTVSS

GLP1R-41-
360
EVQLVESGGGLVQPGGSLRLSCAASGFGIDfyAMGWFRQAPGKEREFVAAISGSG

95

DDTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLEL

FHDEYNVVGQGTLVTVSS

GLP1R-41-
361
EVQLVESGGGLVQPGGSLRLSCAASGNILSLNTMGWFRQAPGKEREFVASVTWG

96

FGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLG

NDWGQGTLVTVSS

GLP1R-41-
362
EVQLVESGGGLVQPGGSLRLSCAASGSIYSLDAMGWFRQAPGKEREFVAAISPAA

97

LTTYVADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAGSSRIYIYSDSLSE

RSYDWGQGTLVTVSS

GLP1R-41-
363
EVQLVESGGGLVQPGGSLRLSCAASGRTFSfYGMGWFRQAPGKERELVAIKFSGG

98

TTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRWD

WGQGTLVTVSS

GLP1R-41-
364
EVQLVESGGGLVQPGGSLRLSCAASGRTFSKYAMGWFRQAPGKEREFVAAIRWS

99

GGTTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGGWGTGRYN

WGQGTLVTVSS

GLP1R-44-
365
EVQLVESGGGLVQPGGSLRLSCAASGSIFSIYAMDWFRQAPGKEREFVAAISSDDS

01

TTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCTAVLPAYDDWGQG

TLVTVSS

GLP1R-44-
366
EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSD

02

GRTYVADSVKGRFT1SADNSKNTAYLQMNSLKPEDTAVYYCAAGLNGAAAAWG

QGTLVTVSS

GLP1R-44-
367
EVQLVESGGGLVQPGGSLRLSCAASGRTFSNGPMGWFRQAPGKEREFVAHISTG

03

GATNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQGWGQ

GTLVTVSS

GLP1R-44-
368
EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSG

04

GTTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRHSYVDWG

QGTLVTVSS

GLP1R-44-
369
EVQLVESGGGLVQPGGSLRLSCAASGSIGSINAMGWFRQAPGKEREFVAAISWSG

05

GATNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASVAYSDYDLG

NDWGQGTLVTVSS

GLP1R-44-
370
EVQLVESGGGLVQPGGSLRLSCAASGLSFDDYAMGWFRQAPGKEREFVAAISGR

06

SGNTYVADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALIQRRAPYSRLE

TWGQGTLVTVSS

GLP1R-44-
371
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMGWFRQAPGKEREGVAAISWS

07

GGTTYVADSVKGRFT1SADNSKNTAYLQMNSLKPEDTAVYYCAAAAGWVAEYG

YWGQGTLVTVSS

GLP1R-44-
372
EVQLVESGGGLVQPGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVATISSNG

08

NTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADLRVLRLRRYE

YNYWGQGTLVTVSS

GLP1R-44-
373
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSNAMGWFRQAPGKEREGVAAISTS

09

GGITYVADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAERDGYGYVVG

QGTLVTVSS

GLP1R-44-
374
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKERELVAGISWN

10

GGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVRAGYDYWG

QGTLVTVSS

GLP1R-44-
375
EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREWVATISWS

11

GGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRSGRDYWG

QGTLVTVSS

GLP1R-44-
376
EVQLVESGGGLVQPGGSLRLSCAASGRAFESYAMGWFRQAPGKEREFVAAIRWS

12

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATGGWGTGRYN

WGQGTLVTVSS

GLP1R-44-
377
EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGD

13

GDSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYWYYTYD

SWGQGTLVTVSS

GLP1R-44-
378
EVQLVESGGGLVQPGGSLRLSCAASGRIFSDYAMGWFRQAPGKEREFVATINGD

14

GDSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANTYCNYTYD

SWGQGTLVTVSS

GLP1R-44-
379
EVQLVESGGGLVQPGGSLRLSCAASGRTLSRSNMGWFRQAPGKEREFVAAVRW

15

SGGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALGPVRRSRLE

WGQGTLVTVSS

GLP1R-44-
380
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKEREFVAAITWS

16

GGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRAGRDSWG

QGTLVTVSS

GLP1R-44-
381
EVQLVESGGGLVQPGGSLRLSCAASGRTFNSYAMGWFRQAPGKEREFVAGITRS

17

AVSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAFRGIMRPDWG

QGTLVTVSS

GLP1R-44-
382
EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVASITWS

18

GGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRGSGRDYW

GQGTLVTVSS

GLP1R-44-
383
EVQLVESGGGLVQPGGSLRLSCAASGRALSSNSMGWFRQAPGKEREFVALITRSG

19

GTTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALNNRRRYVDWG

QGTLVTVSS

GLP1R-44-
384
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS

20

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVGRNGRDYWG

GTLVTVSS

GLP1R-44-
385
EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSG

21

GNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPTIAYNTGYD

YWGQGTLVTVSS

GLP1R-44-
386
EVQLVESGGGLVQPGGSLRLSCAASGRTFDDYAMGWFRQAPGKERELVSGITWS

22

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDGYDY

WGQGTLVTVSS

GLP1R-44-
387
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVSAISTDD

23

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALPDDTYLATT

YDYWGQGTLVTVSS

GLP1R-44-
388
EVQLVESGGGLVQPGGSLRLSCAASGSIFSDNVMGWFRQAPGKEREMVAAIRWS

24

GGITWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDLSGRGDVSE

EYDWGQGTLVTVSS

GLP1R-44-
389
EVQLVESGGGLVQPGGSLRLSCAASGEIASIIAMGWFRQAPGKEREWVSAINSGG

25

DTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADRSRTIWPDWG

QGTLVTVSS

GLP1R-44-
390
EVQLVESGGGLVQPGGSLRLSCAASGRTFSVSTMGWFRQAPGKEREIVAAITWSG

26

SATYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQRRWSQDWGQ

GTLVTVSS

GLP1R-44-
391
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKERELVAGITGG

27

GSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTRYGYDYW

GQGTLVTVSS

GLP1R-44-
392
EVQLVESGGGLVQPGGSLRLSCAASGIPFRSRTMGWFRQAPGKEREFVAGITRNSI

28

RTRYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAPRRPYLPIRIRD

YIWGQGTLVTVSS

GLP1R-44-
393
EVQLVESGGGLVQPGGSLRLSCAASGRTIVPYTMGWFRQAPGKEREFVAAISWS

29

GASTIYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIGGTLYDRRRFE

WGQGTLVTVSS

GLP1R-44-
394
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNNAMGWFRQAPGKEREGVAAINGS

30

GSITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAARDDYGYVVG

QGTLVTVSS

GLP1R-44-
395
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYGMGWFRQAPGKEREGVAGISWS

31

DGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAASDASFDYW

GQGTLVTVSS

GLP1R-44-
396
EVQLVESGGGLVQPGGSLRLSCAASGGTFSDYGMGWFRQAPGKEREGVASISWN

32

DGSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAATADYDYWG

QGTLVTVSS

GLP1R-44-
397
EVQLVESGGGLVQPGGSLRLSCAASGSTFSTYAMGWFRQAPGKERELVAAISWS

33

SGTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLVTSDGVSE

YNYWGQGTLVTVSS

GLP1R-44-
398
EVQLVESGGGLVQPGGSLRLSCAASGFLFDSYAMGWFRQAPGKEREPVAAISPA

34

ALTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAYYTDYDEALE

ETRGSYDWGQGTLVTVSS

GLP1R-44-
399
EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYAMGWFRQAPGKEREGVAAISWN

35

SGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDARRYGYWG

QGTLVTVSS

GLP1R-44-
400
EVQLVESGGGLVQPGGSLRLSCAASGSTFGNYAMGWFRQAPGKEREFVAAISRS

36

GSITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDEDYALGPNE

YDWGQGTLVTVSS

GLP1R-44-
401
EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYAMGWFRQAPGKERELVAGISWG

37

GDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGNGYDYW

GQGTLVTVSS

GLP1R-44-
402
EVQLVESGGGLVQPGGSLRLSCAASGFNSGSYTMGWFRQAPGKEREGVSYISSSD

38

GRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAALDGYSGSWG

QGTLVTVSS

GLP1R-44-
403
EVQLVESGGGLVQPGGSLRLSCAASGLTFWTSGMGWFRQAPGKEREYVAAISRS

39

GSLKGYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATVATALIWGQG

TLVTVSS

GLP1R-44-
404
EVQLVESGGGLVQPGGSLRLSCAASGFTFSINAMGWFRQAPGKERELVSGISWGG

40

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVNEDGFDYWG

QGTLVTVSS

GLP1R-44-
405
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDNAMGWFRQAPGKERELVAAISTS

41

GSNTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAELREYGYWG

QGTLVTVSS

GLP1R-44-
406
EVQLVESGGGLVQPGGSLRLSCAASGRTFTSYNMGWFRQAPGKEREFLGSILWS

42

DDSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASWDGRQVWGQ

GTLVTVSS

GLP1R-44-
407
EVQLVESGGGLVQPGGSLRLSCAASGFTFRNYVMGWFRQAPGKEREFVAAINW

43

NGSITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRSARNYW

GQGTLVTVSS

GLP1R-44-
408
EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISTSG

44

GITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDRIEYSRGGYD

YWGQGTLVTVSS

GLP1R-44-
409
EVQLVESGGGLVQPGGSLRLSCAASGSTFRKYAMGWFRQAPGKEREFVAAISSG

45

GGSTNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGRYRERDSW

GQGTLVTVSS

GLP1R-44-
410
EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISWSG

46

DTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAIDLPDDTYLATE

YDYWGQGTLVTVSS

GLP1R-44-
411
EVQLVESGGGLVQPGGSLRLSCAASGSGFSPNVMGWFRQAPGKERELVAIKFSG

47

GTTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAYEEGVYRW

DWGQGTLVTVSS

GLP1R-44-
412
EVQLVESGGGLVQPGGSLRLSCAASGRTLTNHDMGWFRQAPGKEREGVSYISMS

48

DGRTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLDGYSGSW

GQGTLVTVSS

GLP1R-44-
413
EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKEREFVAAISRSG

49

DSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTLDNYGYVVG

QGTLVTVSS

GLP1R-44-
414
EVQLVESGGGLVQPGGSLRLSCAASGGTASSYHMGWFRQAPGKEREFVAFIHRS

50

GTSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADSITDRRSVA

VAHTSYYWGQGTLVTVSS

GLP1R-44-
415
EVQLVESGGGLVQPGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREIVAAITWS

51

GGITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHGSILLDRIEW

GQGTLVTVSS

GLP1R-44-
416
EVQLVESGGGLVQPGGSLRLSCAASGGTFSIYAMGWFRQAPGKERELVAAISSSG

52

SITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAAAALDGPGDMY

DYWGQGTLVTVSS

GLP1R-44-
417
EVQLVESGGGLVQPGGSLRLSCAASGGIFDNYAMGWFRQAPGKERELVSGINSD

53

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVPISSPSDRNY

WGQGTLVTVSS

GLP1R-44-
418
EVQLVESGGGLVQPGGSLRLSCAASGRTFSLTAMGWFRQAPGKEREFVAAISPAA

54

LTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCASRRAFRLSSDYE

WGQGTLVTVSS

GLP1R-44-
419
EVQLVESGGGLVQPGGSLRLSCAASGRNLRMYRMGWFRQAPGKEREFVAAVN

55

WNGDSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAANWKMLL

GVENDWGQGTLVTVSS

GLP1R-44-
420
EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMGWFRQAPGKERELVAGISSSG

56

GSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGTYDYWGQ

GTLVTVSS

GLP1R-44-
421
EVQLVESGGGLVQPGGSLRLSCAASGRTFDIYAMGWFRQAPGKERELVAAINRD

57

DSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGLGNYNY

WGQGTLVTVSS

GLP1R-44-
422
EVQLVESGGGLVQPGGSLRLSCAASGRSFSFNAMGWFRQAPGKERELVAAITKL

58

GFRNYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAASIEGVSGRWGQ

GTLVTVSS

GLP1R-44-
423
EVQLVESGGGLVQPGGSLRLSCAASGSFFSINAMGWFRQAPGKERELVSASTWN

59

GGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAHRIVVGGTSV

GDWRWGQGTLVTVSS

GLP1R-44-
424
EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYAMGWFRQAPGKEREFVAGITSS

60

GGYTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVVYYGDWE

GSEPVQHEYDWGQGTLVTVSS

GLP1R-44-
425
EVQLVESGGGLVQPGGSLRLSCAASGSIFSRNAMGWFRQAPGKEREFVAAIRWS

61

GKETWYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAKTKRTGIFTTA

RMVDWGQGTLVTVSS

GLP1R-44-
426
EVQLVESGGGLVQPGGSLRLSCAASGGTFDTYAMGWFRQAPGKEREFVAGISGD

62

GTITYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCATDNPYWSGYNY

WGQGTLVTVSS

GLP1R-44-
427
EVQLVESGGGLVQPGGSLRLSCAASGGTFSNYAMGWFRQAPGKERELVSGINSD

63

GGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVSTNDGYDY

WGQGTLVTVSS

GLP1R-44-
428
EVQLVESGGGLVQPGGSLRLSCAASGGIYRVNTMGWFRQAPGKERELVAIKFSG

64

GTTDYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIAHEEGVYRW

DWGQGTLVTVSS

GLP1R-44-
429
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMGWFRQAPGKERELVAGISSSG

65

SSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVSDGGYDYWG

QGTLVTVSS

GLP1R-44-
430
EVQLVESGGGLVQPGGSLRLSCAASGRTSSIYNMGWFRQAPGKEREFVAAISRSG

66

RSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAIVTYSDYDLGN

DWGQGTLVTVSS

GLP1R-44-
431
EVQLVESGGGLVQPGGSLRLSCAASGRALSSYSMGWFRQAPGKEREFVALITRSG

67

GTTFYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCALDNRRSYVDWG

QGTLVTVSS

GLP1R-44-
432
EVQLVESGGGLVQPGGSLRLSCAASGRALSRYGMVWFRQAPGKEREFVAAINRG

68

GKISHYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAGNGGRNYGHS

RARYEWGQGTLVTVSS

GLP1R-44-
433
EVQLVESGGGLVQPGGSLRLSCAASGFKFNDSYMRWFRQAPGKEREFVVAINWS

69

SGSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVNGPIFWGQG

TLVTVSS

GLP1R-44-
434
EVQLVESGGGLVQPGGSLRLSCAASGRTLSDYALGWFRQAPGKERELVSGINTSG

70

DTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAVVTSSYDYWGQ

GTLVTVSS

GLP1R-44-
435
EVQLVESGGGLVQPGGSLRLSCAASGSTFDIYGMGWFRQAPGKEREGVAAITGD

71

GSSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAADNDTEYGYW

GQGTLVTVSS

GLP1R-44-
436
EVQLVESGGGLVQPGGSLRLSCAASGGTLDIYAMGWFRQAPGKEREFVAAISWS

72

GSTTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVLGYDRDYW

GQGTLVTVSS

GLP1R-44-
437
EVQLVESGGGLVQPGGSLRLSCAASGRPYSYDAMGWFRQAPGKEREIVAAISRT

73

GSSIYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAQGSLYDDYDG

LPIKYDWGQGTLVTVSS

GLP1R-44-
438
EVQLVESGGGLVQPGGSLRLSCAASGRTFRTYGMGWFRQAPGKEREGVAAISWS

74

GNSTSYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAARLSKRGNRSS

RDYWGQGTLVTVSS

GLP1R-44-
439
EVQLVESGGGLVQPGGSLRLSCAASGSTFDNYAMGWFRQAPGKERELVAGINWS

75

DSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVAGWGEYDY

WGQGTLVTVSS

GLP1R-44-
440
EVQLVESGGGLVQPGGSLRLSCAASGSTFSIYAMGWFRQAPGKERELVAGINWS

76

DSSTYYADSVKGRFTISADNSKNTAYLQMNSLKPEDTAVYYCAAVTDYDEYNY

WGQGTLVTVSS

TABLE 12

Variable Heavy Chain CDRs

SEQ ID

SEQ ID

SEQ ID

Variant
NO
CDR1
NO
CDR2
NO
CDR3

GLP1R-3
441
GFTFSSYG
620
ISYDESNK
799
AKHMSMQEGAVTGEG

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
442
GFTFSDYG
621
ISYDRSNE
800
AKHMSMQEGAVTGDG

065

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
443
GFTFSDYG
622
ISYDETNK
801
AKHMSMQEGAVTGEG

075

QAAKEFIAWLVKGIVR

ADLVGDAFDV

GLP1R221-
444
GFTFSDYG
623
ISYDESNK
802
AKHMSMQEGAVTGEY

017

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
445
GFTFSDYG
624
ISHDRSNK
803
AKHMSMQEGAVTGEG

033

QAAKDFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
446
GFTFSDYG
625
ISYDETNK
804
AKHMSMQEGAVTGEG

076

QAAKEFIAWLVKGIVR

ADLVGDAFDV

GLP1R221-
447
GFTFSDYG
626
ISYGGSNK
805
AKHMSMQEGAVTGEG

092

QAVKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
448
GFTFSDYG
627
ISHDRSNK
806
AKHMSMQEGAVTGEG

034

QAVKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
449
GFTFSDYG
628
ISYDRSNE
807
AKHMSMQEGAVTGEG

066

QAIKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
450
GFTFSDYG
629
ISSDENNK
808
AKHMSMQEGAVTGEM

084

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
451
GFTFSDYG
630
ISDEGSNK
809
AKHMSMQEGAVTGAG

009

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
452
GFTFSDYG
631
ISSDENNK
810
AKHMSMQEGAVTGEF

072

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
453
GFTFSDYG
632
TSYDESN
811
AKHMSMQEGAVTGEY

044

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
454
GFTFSDYG
633
ISSDASDK
812
AKHMSMQEGAVTGEY

012

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
455
GFTFSDYG
634
TSYDESN
813
AKHMSMQEGAVTGVG

042

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
456
GFTFSDYG
635
ISYEGSNK
814
AKHMSMQEGAVTGMG

051

QAAKEFIAWLIKGRVR

ADLVGDAFDV

GLP1R221-
457
GFTFSDYG
636
ISSDASDK
815
AKHMSMQEGAVTGMG

083

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
458
GFTFSDYG
637
ISYDESNE
816
AKHMSMQEGAVTGEH

040

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
459
GFTFSDYG
638
ISYDRSNE
817
AKHMSMQEGAVHGEG

052

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
460
GFTFSDYG
639
ISDEGSNK
818
AKHMSMQEGAVTGEW

003

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
461
GFTFSDYG
640
ISSDENNK
819
AKHMSMQEGAVTGEF

094

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
462
GFTFSDYG
641
ISYDASNK
820
AKHMSMQEGAVTGEG

001

QAVKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
463
GFTFSDYG
642
ISSDASDK
821
AKHMSMQEGAVTGEW

014

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
464
GFTFSDYG
643
ISHDRSNK
822
AKHMSMQEGAVTGLG

085

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
465
GFTFSDYG
644
ISYDANN
823
AKHMSMQEGAVTGEG

022

K

QAAKEFIAWLIKGRVR

ADLVGDAFDV

GLP1R221-
466
GFTFSDYG
645
ISYEGSNQ
824
AKHMSMQEGAVTGIG

056

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
467
GFTFSDYG
646
TSYDESN
825
AKHMSMQEGAVTGFG

088

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
468
GFTFSDYG
647
ISYDATNK
826
AKHMSMQEGAVTGMG

077

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
469
GFTFSDYG
648
ISYHGSNK
827
AKHMSMQEGAVTGMG

027

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
470
GFTFSDYG
649
ISYDASNK
828
AKHMSMQEGAVTGYG

019

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
471
GFTFSDYG
650
ISSDASDK
829
AKHMSMQEGAVTGEF

029

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
472
GFTFSDYG
651
TSYDESN
830
AKHMSMQEGAVTGGG

043

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
473
GFTFSDYG
652
ISSDASNK
831
AKHMSMQEGAVTGEG

082

QAVKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
474
GFTFSDYG
653
ISYDANN
832
AKHMSMQEGAVTGEW

079

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
475
GFTFSDYG
654
ISHDRSNK
833
AKHMSMQEGAVTGPG

080

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
476
GFTFSDYG
655
IRYGGSNK
834
AKHMSMQEGAVTGEG

059

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
477
GFTFSDYG
656
ISYDATNK
835
AKHMSMQEGAVTGYG

069

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
478
GFTFSDYG
657
ISDEGSNK
836
AKHMSMQEGAVTGMG

036

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
479
GFTFSDYG
658
ISYEGSNQ
837
AKHMSMQEGAVTGWG

057

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
480
GFTFSDYG
659
ISDEGSNK
838
AKHMSMQEGAVTGLG

035

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
481
GFTFSDYG
660
ISDEGSNK
839
AKHMSMQEGAVTGEW

063

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
482
GFTFSDYG
661
TSYDESN
840
AKHMSMQEGAVTGEW

090

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
483
GFTFSDYG
662
ISSDASHK
841
AKHMSMQEGAVTWEG

002

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
484
GFTFSDYG
663
ISYDETNK
842
AKHMSMQEGAVTGFG

087

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
485
GFTFSDYG
664
ISDEGSNK
843
AKHMSMQEGAVTGMG

038

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
486
GFTFSDYG
665
ISYGGSNK
844
AKHMSMQEGAVTNEG

060

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
487
GFTFSDYG
666
ISSDASHK
845
AKHMSMQEGAVTWEG

015

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
488
GFTFSDYG
667
ISYDESNK
846
AKHMSMQEGAVTGEW

020

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
489
GFTFSDYG
668
ISSDASDK
847
AKHMSMQEGAVTGGG

011

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
490
GFTFSDYG
669
ISYGGSNK
848
AKHMSMQEGAVTGEW

091

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
491
GFTFSDYG
670
TSYDESN
849
AKHMSMQEGAVTGEW

086

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
492
GFTFSDYG
671
ISHDRSNK
850
AKHMSMQEGAVTGEG

074

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
493
GFTFSDYG
672
ISHDRSNK
851
AKHMSMQEGAVTGEG

032

QAAKDFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
494
GFTFSDYG
673
ISSDASDK
852
AKHMSMQEGAVTGWG

013

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
495
GFTFSDYG
674
ISHDRSNK
853
AKHMSMQEGAVTGWG

058

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
496
GFTFSDYG
675
ISSDASDK
854
AKHMSMQEGAVTGEG

031

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
497
GFTFSDYG
676
ISSDASDK
855
AKHMSMQEGAVTGEG

054

WAAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R221-
498
GFTFSDYG
677
ISYDATNK
856
AKHMSMQEGAVTGEG

021

QFAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
499
GFTFSDYG
678
ISSDASHK
857
AKHMSMQEGAVTWEG

016

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
500
GFTFSDYG
679
ISSDASDK
858
AKHMSMQEGAVTGEG

030

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
501
GFTFSDYG
680
ISSDASDK
859
AKHMSMQEGAVTGEW

018

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
502
GFTFSDYG
681
ISYDAGN
860
AKHMSMQEGAVTGMG

028

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
503
GFTFSDYG
682
TSYEESNK
861
AKHMSMQEGAVTGMG

023

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
504
GFTFSDYG
683
ISHDRSNK
862
AKHMSMQEGAVTGIG

089

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
505
GFTFSDYG
684
ISSDASDK
863
AKHMSMQEGAVTGWG

053

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
506
GFTFSDYG
685
ISSDENNK
864
AKHMSMQEGAVTGIG

071

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
507
GFTFSDYG
686
ISYGGSNK
865
AKHMSMQEGAVTGWG

055

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
508
GFTFSDYG
687
ISSDASNK
866
AKHMSMQEGAVTGMG

046

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
509
GFTFSDYG
688
IRYDESNK
867
AKHMSMQEGAVTGEG

039

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
510
GFTFSDYG
689
ISSDASNK
868
AKHMSMQEGAVMGEG

078

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
511
GFTFSDYG
690
ISSDASDK
869
AKHMSMQEGAVTGIG

010

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
512
GFTFSDYG
691
ISDEGSNK
870
AKHMSMQEGAVTGLG

005

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
513
GFTFSDYG
692
ISHDRSNK
871
AKHMSMQEGAVTGFG

073

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
514
GFTFSDYG
693
ISYDETNK
872
AKHMSMQEGAVTGIG

041

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
515
GFTFSDYG
694
ISYDESNK
873
AKHMSMQEGAVTEEG

025

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
516
GFTFSDYG
695
ISDEGSNK
874
AKHMSMQEGAVTGWG

007

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
517
GFTFSDYG
696
ISYDESNK
875
AKHMSMQEGAVTGFG

093

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
518
GFTFSDYG
697
ISYDAGN
876
AKHMSMQEGAVTGEG

024

K

QAVKEFIAWLVKGDVR

ADLVGDAFDV

GLP1R221-
519
GFTFSDYG
698
ISDEGSNK
877
AKHMSMQEGAVTGLG

008

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
520
GFTFSDYG
699
ISYDENNK
878
AKHMSMQEGAVTGMG

050

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
521
GFTFSDYG
700
TSYDESN
879
AKHMSMQEGAVTGWG

062

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
522
GFTFSDYG
701
ISYDAGN
880
AKHMSMQEGAVTGFG

068

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
523
GFTFSDYG
702
ISNDENNK
881
AKHMSMQEGAVTGFG

067

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
524
GFTFSDYG
703
TSYDESN
882
AKHMSMQEGAVTGWG

061

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
525
GFTFSDYG
704
ISDEGSNK
883
AKHMSMQEGAVTGYG

064

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
526
GFTFSDYG
705
ISYDATNK
884
AKHMSMQEGAVTGIG

070

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
527
GFTFSDYG
706
ISDEGSNK
885
AKHMSMQEGAVTGFG

006

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
528
GFTFSDYG
707
ISSDASNK
886
AKHMSMQEGAVTGEG

045

QAAKEFIAWLVFGRVR

ADLVGDAFDV

GLP1R221-
529
GFTFSDYG
708
ISDEGSNK
887
AKHMSMQEGAVTGFG

004

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R221-
530
GFTFSDYG
709
ISSDASDK
888
AKHMSMQEGAVTGEG

047

QAWKEFIAWLVKGRV

RADLVGDAFDV

GLP1R221-
531
GFTFSDYG
710
ISSDASDK
889
AKHMSMQEGAVTGEY

048

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
532
GFTFNNYP
711
ISYDESNK
890
AKHMSMQEGAVTGGG

052

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
533
GFTFNNY
712
ISDEGSNK
891
AKHMSMQEGAVTGEY

016

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
534
GFSFSDYG
713
ISYDANN
892
AKHMSMQEGAVTGEW

023

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
535
GFAFSNY
714
ISYDESNK
893
AKHMSMQEGAVTGEW

014

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
536
GFSFSDYG
715
ISYEGSNK
894
AKHMSMQEGAVTGEK

090

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
537
GFTFRDY
716
IRYDEINK
895
AKHMSMQEGAVTGEG

073

G

QAAKEFIAWLVGGRVR

ADLVGDAFDV

GLP1R-222-
538
GFTFNNY
717
ISDEGSNK
896
AKHMSMQEGAVTGVG

012

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
539
GFTFSAYS
718
ISYDATNK
897
AKHMSMQEGAVTGEF

082

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
540
GFTFDNY
719
ISYDAGN
898
AKHMSMQEGAVTGEG

081

A

K

QAAKEFIAWLVKGFVR

ADLVGDAFDV

GLP1R-222-
541
GFPFSSYA
720
ISYDRSNK
899
AKHMSMQEGAVTGYG

056

QAAKEFIAWLVKGFVR

ADLVGDAFDV

GLP1R-222-
542
GFTFRDY
721
ISFDESNK
900
AKHMSMQEGAVTGEW

058

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
543
GFTFNNYP
722
ISHDRSNK
901
AKHMSMQEGAVTGTG

063

QAAKEFIAWLVKGIVR

ADLVGDAFDV

GLP1R-222-
544
GLTFSNY
723
TSYDESN
902
AKHMSMQEGAVTREG

042

A

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
545
GFTFGSYA
724
TSYDESN
903
AKHMSMQEGAVTGEG

092

K

QAAKEFIAWLVMGRVR

ADLVGDAFDV

GLP1R-222-
546
GFTFSSYG
725
ISSDASDK
904
AKHMSMQEGAVTGEG

007

QAAKEFIAWLVKGWV

RADLVGDAFDV

GLP1R-222-
547
GFNFNNY
726
ISYDASNK
905
AKHMSMQEGAVTGEF

008

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
548
GFTSSSYA
727
ISDEGSNK
906
AKHMSMQEGAVTGEG

024

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
549
GFTFSDYP
728
ISYDESNK
907
AKHMSMQEGAVTGEG

062

QAAKEFIAWLVKGRVR

NDLVGDAFDV

GLP1R-222-
550
GFTFGNY
729
ISYDASNK
908
AKHMSMQEGAVTGEF

077

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
551
GFTFNNY
730
ISYAGSNE
909
AKHMSMQEGAVTGEG

064

A

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
552
GFSFRSYG
731
ISSDASNK
910
AKHMSMQEGAQTGEG

074

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
553
GFSFSNYA
732
TSYDESN
911
AKHMSMQEGAVTGEG

029

K

QAAKEFIAWLLKGRVR

ADLVGDAFDV

GLP1R-222-
554
GFAFSSYA
733
ISYDENNK
912
AKHMSMQEGAVTGYG

046

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
555
GFTFNNYP
734
IWSDASQ
913
AKHMSMQEGAVTGEG

005

K

WAAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
556
GFTFGNY
735
ISSDASDK
914
AKHMSMQEGAVTGEW

004

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
557
GFAFSNY
736
ISYDASNK
915
AKHMSMQEGAVTGEG

022

G

QAAKNFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
558
GFTFSNYA
737
ISYDASNK
916
AKHMSMQEGAVTGYG

087

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
559
GFSFGSYA
738
TSYDESN
917
AKHMSMQEGAVTGEW

048

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
560
GFTFSSYP
739
ISYEGTNK
918
AKHMSMQEGAVTGEG

072

QAAKDFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
561
GFSFSNYA
740
ISYDESNE
919
AKHMSMQEGAVTGEG

089

QAAKEFIAWLVKGDVR

ADLVGDAFDV

GLP1R-222-
562
GFSFSSYG
741
ISYGGSNK
920
AKHMSMQEGAVTGEW

083

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
563
GFSFSNYA
742
TSYDESN
921
AKHMSMQEGAVTGEG

001

K

QAAKEFIAWLLKGRVR

ADLVGDAFDV

GLP1R-222-
564
GFTFSDYG
743
ISYDESNK
922
AKHMSMQEGAVTGEG

075

WAAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
565
GFTFSDFA
744
ISYEGSNK
923
AKHMSMQEGAVQGEG

071

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
566
GFTFSDYP
745
ISDEGSNK
924
AKHMSMQEGAVTGEIQ

069

AAKEFIAWLVKGRVRA

DLVGDAFDV

GLP1R-222-
567
GFTFRDY
746
ISYDATNK
925
AKHMSMQEGAVTGMG

002

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
568
GFTFNRY
747
ISYDASNK
926
AKHMSMQEGAVTGEG

006

G

QAAWEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
569
GFPFSSYG
748
ISYDATNK
927
AKHMSMQEGAVTGEG

055

QAAKSFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
570
GFSFGSYA
749
ISYDASNK
928
AKHMSMQEGAVTGMG

027

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
571
GFTFSNYD
750
ISYAGSNK
929
AKHMSMQEGAVTGTG

066

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
572
GFSFRTYG
751
ISDEGSNK
930
AKHMSMQEGAVTGEG

015

YAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
573
GFTFSTYG
752
ISYDANN
931
AKHMSMQEGAVTGEG

076

K

QAAVEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
574
GFSFSDYA
753
ISSDASNK
932
AKHMSMQEGAVTGYG

011

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
575
GFTFSNYA
754
ISYDATNK
933
AKHMSMQEGAVTGEA

065

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
576
GFTFSNYD
755
TSYDESK
934
AKHMSMQEGAVTGKG

041

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
577
GFSFSNYA
756
TSYDESN
935
AKHMSMQEGAVTGEG

028

K

QAAYEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
578
GFTFSDYP
757
ISYAGSNE
936
AKHMSMQEGAVTGYG

086

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
579
GFPFSSYA
758
ISYDANN
937
AKHMSMQEGAVTGYG

033

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
580
GFAFSSYA
759
ISYDESNK
938
AKHMSMQEGAVTGEG

035

WAAKEFIFWLVKGRVR

ADLVGDAFDV

GLP1R-222-
581
GFSFSNYA
760
ISFDESNK
939
AKHMSMQEGAVTGYG

045

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
582
GFTFSDYP
761
ISYDRSNE
940
AKHMSMQEGAVTGTG

085

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
583
GFSFSNYG
762
ISSDASNK
941
AKHMSMQEGAVTGEW

049

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
584
GFSFRNY
763
ISYDESNK
942
AKHMSMQEGAVTGEG

078

G

QAAKEFIAWLVKGRVR

PDLVGDAFDV

GLP1R-222-
585
GFTFNDY
764
ISSDASNK
943
AKHMSMQEGAVTGTG

021

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
586
GFTFGNY
765
ISSDASNK
944
AKHMSMQEGAVTGEF

009

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
587
GFTFTNY
766
ISSDASDK
945
AKHMSMQEGAVTGMG

036

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
588
GFSFSNYG
767
ISYGGSNK
946
AKHMSMQEGAVTGEG

084

FAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
589
GFTFSDYP
768
ISSDASDK
947
AKHMSMQEGAVTGEG

010

QAAKEFIAWLVKGWV

RADLVGDAFDV

GLP1R-222-
590
GFSFSNYA
769
ISYDASNK
948
AKHMSMQEGAVTGGG

088

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
591
GFPFSNYA
770
ISSDASNK
949
AKHMSMQEGAVTGEW

079

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
592
GFSFSDYG
771
ISYDANN
950
AKHMSMQEGAVTGLG

040

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
593
GFTFGSYG
772
ISDEGSNK
951
AKHMSMQEGAVTNEG

070

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
594
GFTFNDY
773
ISSDENNK
952
AKHMSMQEGAVTGEG

032

G

QWAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
595
GFTFRDY
774
ISSDENNK
953
AKHMSMQEGAVTGWG

030

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
596
GFTFGNY
775
ISSDASHK
954
AKHMSMQEGAVTWEG

038

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
597
GFTFSGYA
776
ISSDENNK
955
AKHMSMQEGAVTGWG

031

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
598
GFTFSNYA
777
ISDEGSNK
956
AKHMSMQEGAVTGAG

026

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
599
GFNFNNY
778
ISYDESNK
957
AKHMSMQEGAVTGEW

054

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
600
GFTFSDYP
779
ISSDASDK
958
AKHMSMQEGAVTGHG

093

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
601
GFTFNNYP
780
ISYGGSDK
959
AKHMSMQEGAVTGEG

051

WAAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
602
GFTFSDYA
781
IPYDESNK
960
AKHMSMQEGAVTGEG

067

QAAKNFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
603
GFAFSNY
782
ISDEGSNK
961
AKHMSMQEGAVTGHG

059

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
604
GFTFNRY
783
ISDEGSNK
962
AKHMSMQEGAVTGVG

025

G

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
605
GFIFSNYA
784
ISYDASNK
963
AKHMSMQEGAVTGEY

068

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
606
GFNFNNY
785
ISSDASNK
964
AKHMSMQEGAVTGEG

053

G

QAVKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
607
GFTFGSYG
786
ISSDENNK
965
AKHMSMQEGAVTGEG

018

FAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
608
GFTFGSYA
787
TSYDESN
966
AKHMSMQEGAVTGYG

047

K

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
609
GFTFSNYD
788
ISDEGSNK
967
AKHMSMQEGAVTGEG

060

WAAKEFIAWLVKGRV

RADLVGDAFDV

GLP1R-222-
610
GFTFKNY
789
ISYGGSNK
968
AKHMSMQEGAVTGEG

020

G

PAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
611
GFSFSDYA
790
ISDDGSNK
969
AKHMSMQEGAVTGEG

044

QAAKEFIAWLVKGFVR

ADLVGDAFDV

GLP1R-222-
612
GFSFSDYG
791
ISSDASDK
970
AKHMSMQEGAVTGEG

080

QALKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
613
GFTFGSYG
792
ISSDENNK
971
AKHMSMQEGAVTGMG

057

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
614
GFTLSNY
793
IPYDESNK
972
AKHMSMQEGAVTGVG

043

A

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
615
GFTFSNFA
794
ISSDASNK
973
AKHMSMQEGAVTGEG

003

QSAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
616
GFTFRNFG
795
ISSDASNK
974
AKHMSMQEGAVTGIG

037

QAAKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
617
GFTFGSHG
796
ISSDENNK
975
AKHMSMQEGAVTGEG

091

QAIKEFIAWLVKGRVR

ADLVGDAFDV

GLP1R-222-
618
GFNFNNY
797
ISDEGSNK
976
AKHMSMQEGAVTGEG

019

G

QAAKEFIAWLVKGRVR

PDLVGDAFDV

GLP1R-222-
619
GFTFGSYG
798
ISYDASNK
977
AKHMSMQEGAVTGWG

094

QAAKEFIAWLVKGRVR

ADLVGDAFDV

TABLE 13

Variable Light Chain CDRs

SEQ ID

SEQ ID

SEQ ID

Variant
NO
CDR1
NO
CDR2
NO
CDR3

GLP1R-3
978
SSNIADNY
1157
DNN
1336
GTWDNYLGAGV

GLP1R221-065
979
TSNIANNF
1158
DHN
1337
GTWDTSLSAGA

GLP1R221-075
980
GSNIGNND
1159
DND
1338
GTWDTSLSNYV

GLP1R221-017
981
SSNIGNTY
1160
DDY
1339
ATWDATLNTGV

GLP1R221-033
982
SSNIGNEY
1161
DNN
1340
ATWDTSLNVGV

GLP1R221-076
983
SSNIGNND
1162
ENN
1341
LTWDHSLTAYV

GLP1R221-092
984
TSNIANNF
1163
DNN
1342
GTWDTSLSVGM

GLP1R221-034
985
SSNIGNNP
1164
END
1343
ATWDRGLSTGV

GLP1R221-066
986
SSNIGNNY
1165
ENN
1344
GIWDRSLSAWV

GLP1R221-084
987
SSNIADNY
1166
ENN
1345
GTWDVSLSVGM

GLP1R221-009
988
SSNIGNQY
1167
DDH
1346
GTWDTSLSVGE

GLP1R221-072
989
SSNIGRNF
1168
DHN
1347
GTWDVTLHTGV

GLP1R221-044
990
SSNIGNND
1169
DNN
1515
GTWDTSLSGGV

GLP1R221-012
991
SSTIGNNY
1170
EDD
1516
ATWDRGLSTGV

GLP1R221-042
992
SSNIGNKY
1171
DDD
1517
GTWDTSLSVGM

GLP1R221-051
993
SSNIGNDY
1172
DNN
1518
GTWDRGPNTGV

GLP1R221-083
994
SSNIGSKD
1173
DDD
1519
GTWDRSLGGWV

GLP1R221-040
995
SSNIGDND
1174
DNN
1353
GTWDRSLNVGV

GLP1R221-052
996
SSNIGSKY
1175
DNN
1354
GTWDRGPNTGV

GLP1R221-003
997
SSNIGNNP
1176
DND
1355
ATWDHSLRVGV

GLP1R221-094
998
SSNIGNKY
1177
DNN
1356
GTWDTALTAGV

GLP1R221-001
999
SSNIGSHY
1178
DTN
1357
ATWDRGLSTGV

GLP1R221-014
1000
SSTIGNNY
1179
DND
1358
ATWDTSLNVGV

GLP1R221-085
1001
TSNIGNNH
1180
DNN
1359
GTWDRSLSSAV

GLP1R221-022
1002
SSNIGSNY
1181
DNN
1360
GTWDTSVSAGV

GLP1R221-056
1003
GSNIGNND
1182
DTN
1361
ATWDRTLSIGV

GLP1R221-088
1004
SSNIGSKY
1183
DNN
1362
GTWDTTLNIGV

GLP1R221-077
1005
SSNIGNND
1184
GDD
1363
ATWDRSLRAGV

GLP1R221-027
1006
SSNIGNDF
1185
DNN
1364
GTWDTSLSTGV

GLP1R221-019
1007
SSNIGNNF
1186
DNN
1365
GTWETSLSAGV

GLP1R221-029
1008
SSNIGNND
1187
EDN
1366
GTWVTSLSAGV

GLP1R221-043
1009
SSNIGNHD
1188
DNN
1367
GTWDRSLSGEV

GLP1R221-082
1010
SSNIGSNF
1189
DDK
1368
ATWDRGLSTGV

GLP1R221-079
1011
SSNIGDND
1190
DND
1369
ATWDRSLSAVV

GLP1R221-080
1012
SSNIGNND
1191
DDD
1370
GTWDKSLSAVV

GLP1R221-059
1013
SSNIGDND
1192
ENN
1371
GTWDTSLSGGV

GLP1R221-069
1014
SSNIGKNF
1193
DNN
1372
GTWDVTLHTGV

GLP1R221-036
1015
SSNIGNEY
1194
ENK
1373
GTWDASLSAGL

GLP1R221-057
1016
SSNIGSKY
1195
DNN
1374
GTWESSLSAGV

GLP1R221-035
1017
SSDIGNKY
1196
ENN
1375
ATWDASLSGGV

GLP1R221-063
1018
SSNIGNNF
1197
ENN
1376
ATWDATLNTGV

GLP1R221-090
1019
SSNIGSNY
1198
DTD
1377
GTWDVSLNTQV

GLP1R221-002
1020
SSNIGNKY
1199
DTN
1378
ATWDATLNTGV

GLP1R221-087
1021
SSNIGKDY
1200
ENV
1379
GTWDASLSGVV

GLP1R221-038
1022
TSNIGNND
1201
DNN
1380
GTWDVTLHTGV

GLP1R221-060
1023
GSNIGNND
1202
ETN
1381
GTWDTGLSAGV

GLP1R221-015
1024
TSNIGNNY
1203
DTN
1382
ATWDATLNTGV

GLP1R221-020
1025
SSNIGRNF
1204
DNN
1383
GTWDTSLSRYV

GLP1R221-011
1026
SSNIGKDY
1205
DNY
1384
GTWDTSLSVGV

GLP1R221-091
1027
SSNIGSND
1206
VND
1385
GAWDRSLSAYV

GLP1R221-086
1028
SSNIGKHY
1207
DVD
1386
ATWDRGLSTGV

GLP1R221-074
1029
SSNIGSNY
1208
DNN
1387
GTWDTRLSVGV

GLP1R221-032
1030
SSNIGNNY
1209
DNN
1388
ATWDRSLRAGV

GLP1R221-013
1031
SSNIGNKY
1210
DDD
1389
ATWDTSLNVGV

GLP1R221-058
1032
SSNIGKYY
1211
DNN
1390
GTWDTSLATGL

GLP1R221-031
1033
SSNIGSNL
1212
DNN
1391
GTWDTSLSAGA

GLP1R221-054
1034
RSNIGNYY
1213
DHN
1392
ATWDRTLSIGV

GLP1R221-021
1035
SSNIGNNF
1214
DNN
1393
GAWDRSLSAGV

GLP1R221-016
1036
SSNIGNKY
1215
DND
1394
ATWDATLNTGV

GLP1R221-030
1037
SSNIENND
1216
ENN
1395
GTWDRSLSAAL

GLP1R221-018
1038
SSNIGSNH
1217
ENT
1396
ATWDATLNTGV

GLP1R221-028
1039
SSTIGNNY
1218
DND
1397
GTWDKSLSAGV

GLP1R221-023
1040
SSNIGSKD
1219
DTN
1398
ATWDRGLSTGV

GLP1R221-089
1041
SSNIGKDF
1220
DND
1399
ATWDTSLSAEV

GLP1R221-053
1042
SSNIGKDY
1221
EDN
1400
ATWDRTLSIGV

GLP1R221-071
1043
SSNIGSNY
1222
DDN
1401
GTWGSSLSAGL

GLP1R221-055
1044
SSNIGSND
1223
DKN
1402
GAWDRSLSAGV

GLP1R221-046
1045
SSNIGNND
1224
DDD
1403
AAWDDYLSAVV

GLP1R221-039
1046
SSNIGNHF
1225
DNN
1404
GTWDRSLNVGV

GLP1R221-078
1047
SSNIGNNP
1226
ENI
1405
ATWDRSLRAGV

GLP1R221-010
1048
SSTIGNNY
1227
DNN
1406
GTWDASLSVWV

GLP1R221-005
1049
SSTIGNNY
1228
ENR
1407
GTWDNYLGAGV

GLP1R221-073
1050
SSNIGSNH
1229
END
1408
GTWDTSLSAYI

GLP1R221-041
1051
SSNIGSKY
1230
NDN
1409
GTWDTSLSVGM

GLP1R221-025
1052
SSNIGKYY
1231
DNY
1410
ATWDRGLSTGV

GLP1R221-007
1053
SSNIGNND
1232
ENT
1411
GTWDANLRAGV

GLP1R221-093
1054
SSNIENNH
1233
END
1412
ATWDTSLSEGV

GLP1R221-024
1055
SSNIGKYY
1234
DTN
1413
ATWDRGLSTGV

GLP1R221-008
1056
SSSIGNNY
1235
ANN
1414
GTWDISLSAAV

GLP1R221-050
1057
SSNIGNNF
1236
DKN
1415
ATWDTRLSAVV

GLP1R221-062
1058
SSNIGNNY
1237
ENN
1416
GTWDASLGAWV

GLP1R221-068
1059
SSNIGSND
1238
NNN
1417
GTWDARLGGAV

GLP1R221-067
1060
SSNIGNNY
1239
ANN
1418
GTWDARLGGAV

GLP1R221-061
1061
SSNIGTNF
1240
DNN
1419
GTWDNRLSGWV

GLP1R221-064
1062
SSNIGKDY
1241
ENT
1420
ATWDATLNTGV

GLP1R221-070
1063
SSNIENNH
1242
QNN
1421
GTWDVSLNTQV

GLP1R221-006
1064
SSNIGNNH
1243
GSN
1422
GTWDTSLNIGV

GLP1R221-045
1065
SSNIGNND
1244
GNN
1423
GTWDTSLSGGI

GLP1R221-004
1066
SSTIGNNY
1245
DND
1424
GTWESSLSAGV

GLP1R221-047
1067
SSNIGNEY
1246
GDD
1425
GTWDTSLSVGM

GLP1R221-048
1068
SSNIGNHD
1247
AND
1426
GTWDTSLSVGE

GLP1R-222-052
1069
SSNIGKRS
1248
DNN
1427
VTWDRSLSAGV

GLP1R-222-016
1070
SSNIENND
1249
DFN
1428
GTWDTSLSVGM

GLP1R-222-023
1071
SSNIGNND
1250
ENT
1429
GTWDAGLSTGV

GLP1R-222-014
1072
SSNIGNHD
1251
DNN
1430
GTWDTSLSAGV

GLP1R-222-090
1073
SSNIADNY
1252
DNN
1431
ATWDNRLSAGV

GLP1R-222-073
1074
GSNIGNND
1253
DNN
1432
GTWDRGPNTGV

GLP1R-222-012
1075
SSNIGNND
1254
DDD
1433
GTWDTSLSVGE

GLP1R-222-082
1076
SSNIGSKY
1255
DNN
1434
GTWDISPSAWV

GLP1R-222-081
1077
SSNIGSDY
1256
DNN
1435
GTWDESLRSWV

GLP1R-222-056
1078
SSNIGSNY
1257
DND
1436
GTWDTSLSVGE

GLP1R-222-058
1079
SSNIGNNP
1258
DNN
1437
ATWDNKLTSGV

GLP1R-222-063
1080
SSNIGNYY
1259
DNN
1438
ATWDTSLNVGV

GLP1R-222-042
1081
SSNIGNND
1260
DDN
1439
GTWDTSLSAYI

GLP1R-222-092
1082
SSNIGSDY
1261
ENN
1440
GTWDRGPNTGV

GLP1R-222-007
1083
SSDIGNKY
1262
ENN
1441
GTWDTSLSAGA

GLP1R-222-008
1084
SSNIGSNH
1263
DNN
1442
GTWDTSLSVGE

GLP1R-222-024
1085
TSNIGSNF
1264
DEN
1443
ATWDATLNTGV

GLP1R-222-062
1086
SSNIENND
1265
DNN
1444
GTWDRSLNVGV

GLP1R-222-077
1087
SSSIGNNY
1266
ENN
1445
GTWDNNLGAGV

GLP1R-222-064
1088
SSNIGSKY
1267
DDN
1446
GTWDTSLSTGV

GLP1R-222-074
1089
SSNIGNND
1268
DNN
1447
GTWDRGPNTGV

GLP1R-222-029
1090
SSNIGNNY
1269
END
1448
GTWDTSLATGL

GLP1R-222-046
1091
TSNIGNNY
1270
ENT
1449
GTWDTTLSAGV

GLP1R-222-005
1092
SSNIGNDY
1271
DNN
1450
GTWDASLSAGL

GLP1R-222-004
1093
SSNIGNDY
1272
ENN
1451
GTWDASLSAGL

GLP1R-222-022
1094
SSNIGNND
1273
DND
1452
GTWDRTLSIGV

GLP1R-222-087
1095
SSNIENND
1274
DNN
1453
GTWDRRLSDVV

GLP1R-222-048
1096
RSNIGNNF
1275
DNN
1454
GTWDRSLSSAV

GLP1R-222-072
1097
SSSIGNNY
1276
DTN
1455
GTWDRSLNVGV

GLP1R-222-089
1098
SSNIGNND
1277
DTN
1456
GTWDISLSARV

GLP1R-222-083
1099
SSNIGSKY
1278
DND
1457
ATWDTSLSAGV

GLP1R-222-001
1100
SSNIGSKY
1279
DNN
1458
GTWDTSLATGL

GLP1R-222-075
1101
SSNIGSKD
1280
DTY
1459
GTWDTSVSAGV

GLP1R-222-071
1102
TSNIGNNY
1281
DDN
1460
GTWDRSLNVGV

GLP1R-222-069
1103
SSNIGSHY
1282
DNN
1461
GTWHSSLSAGV

GLP1R-222-002
1104
SSDIGNKY
1283
DND
1462
GTWDTTLSAGV

GLP1R-222-006
1105
SSNIGNND
1284
DNN
1463
GAWDTSLSAVV

GLP1R-222-055
1106
TSNIGNNY
1285
DNN
1464
GTWDTSLSVGE

GLP1R-222-027
1107
TSNIGNNH
1286
EDN
1465
GTWDTSLATGL

GLP1R-222-066
1108
SSTIGNNY
1287
DNN
1466
ATWDRGLSTGV

GLP1R-222-015
1109
RSNIGNYY
1288
DND
1467
GTWDRSLSVGL

GLP1R-222-076
1110
SSNIGSKY
1289
DTY
1468
GTWDAGLSTGV

GLP1R-222-011
1111
SSNIGSNY
1290
ENN
1469
GTWDTSLSVGE

GLP1R-222-065
1112
SSTIGNNY
1291
DNN
1470
ATWDRTLSIGV

GLP1R-222-041
1113
SSNIGSKD
1292
DDN
1471
GIWDRSLSAWV

GLP1R-222-028
1114
TSNIGNNH
1293
DNN
1472
GTWDTSLATGL

GLP1R-222-086
1115
SSNIGNHF
1294
DTN
1473
GTWDRGPNTGV

GLP1R-222-033
1116
SSNIGKYY
1295
DNN
1474
GTWDVSLSVGM

GLP1R-222-035
1117
SSNIGNND
1296
ENN
1475
GTWDVSLSVGM

GLP1R-222-045
1118
SSNIGNTY
1297
ENR
1476
ATWDTSLSEGV

GLP1R-222-085
1119
SSNIGSDY
1298
ANN
1477
GTWDVTLHAGV

GLP1R-222-049
1120
TSNIGKNF
1299
ENK
1478
ATWDRSLSAGV

GLP1R-222-078
1121
SSNIGKYY
1300
DTN
1479
GTWDNNLGAGV

GLP1R-222-021
1122
SSNIGDND
1301
ENR
1480
GTWDASLSAGL

GLP1R-222-009
1123
SSNIGKNF
1302
DTN
1481
GTWDTSLSVGE

GLP1R-222-036
1124
SSNIGSKY
1303
DNN
1482
ATWDDTLTAGV

GLP1R-222-084
1125
SSNIGSKD
1304
DNN
1483
GIWDTSLSAWV

GLP1R-222-010
1126
SSNIGNKY
1305
DNN
1484
GTWDNRLSAGV

GLP1R-222-088
1127
SSNIGNNF
1306
DND
1485
GTWDTSLRVVV

GLP1R-222-079
1128
SSNIGSND
1307
NNN
1486
GTWESGLSAGV

GLP1R-222-040
1129
SSNIGNQY
1308
DTY
1487
ATWDHSLRVGV

GLP1R-222-070
1130
SSNIGNND
1309
ANN
1488
GTWHSSLSAGV

GLP1R-222-032
1131
SSNIGNNP
1310
END
1489
GTWDTRLSVGV

GLP1R-222-030
1132
SSNIGNNL
1311
DND
1490
GTWDTSLTAGV

GLP1R-222-038
1133
SSNIGNKY
1312
DTN
1491
ATWDATLNTGV

GLP1R-222-031
1134
SSNIGNNY
1313
DDN
1492
GTWDTSLSVGM

GLP1R-222-026
1135
SSNIGSKY
1314
DNN
1493
GTWDRGPNTGV

GLP1R-222-054
1136
SSNIGSKY
1315
DDY
1494
GTWDNRLSGWV

GLP1R-222-093
1137
RSNIGNNF
1316
DNY
1495
ATWDRGLSTGV

GLP1R-222-051
1138
RSNIGNNF
1317
DNN
1496
ATWDRSLSAGV

GLP1R-222-067
1139
RSNIGNNF
1318
DNN
1497
GTWDRRLSAVV

GLP1R-222-059
1140
SSNIGNEY
1319
ENN
1498
GTWDNYLGAVV

GLP1R-222-025
1141
SSNIGNEY
1320
DND
1499
ATWDATLNTGV

GLP1R-222-068
1142
RSNIGNNF
1321
ENN
1500
GSWDRSLSAVV

GLP1R-222-053
1143
SSNIGNND
1322
ASN
1501
ATWDNILSAWV

GLP1R-222-018
1144
SSNIGKNF
1323
ETN
1502
ATWDRGLSTGV

GLP1R-222-047
1145
SSNIGTNF
1324
ADN
1503
GTWDRTLSIGV

GLP1R-222-060
1146
SSNIGNNP
1325
GNN
1504
GTWDASLGAVV

GLP1R-222-020
1147
SSNIGNND
1326
DND
1505
GTWDAGLSTGV

GLP1R-222-044
1148
SSNIGNNH
1327
DFN
1506
ATWDRSLRAGV

GLP1R-222-080
1149
SSNIGNHD
1328
ENK
1507
GTWESGLSAGV

GLP1R-222-057
1150
SSNIGDHY
1329
ENN
1508
ATWDNKLTSGV

GLP1R-222-043
1151
SSNIGNNY
1330
DNN
1509
ATWDRSLRAGV

GLP1R-222-003
1152
SSNIGNHD
1331
ENN
1510
GTWDTSLSAGV

GLP1R-222-037
1153
SSNIGNNP
1332
NNN
1511
ATWDTTLNTGV

GLP1R-222-091
1154
SSNIGSNY
1333
GND
1512
ASWDNRLTAVV

GLP1R-222-019
1155
SSNIGNNY
1334
DNN
1513
ATWDRGLSTGV

GLP1R-222-094
1156
SSNIGNTY
1335
ENK
1514
ATWDTSLSEGV

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

	Number	Date	Country
	63070734	Aug 2020	US
	63081801	Sep 2020	US

METHODS AND COMPOSITIONS RELATING TO GLP1R VARIANTS

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CROSS-REFERENCE

Provisional Applications (2)