Patent Application
20250011426
The content of the electronically submitted sequence listing (Name: IOTS-100-US-NP Sequence Listing.xml; Size: 16,305 bytes; and Date of Creation: Apr. 2, 2024), filed with the application, is incorporated herein by reference in its entirety.
The present disclosure relates to multispecific antibodies containing a lambda charge pair introduced into the interface of a heavy chain and a light chain as a strategy for reducing chain mispairing. The disclosure also relates to methods of producing these multispecific antibodies and their therapeutic uses.
Multispecific antibodies, which recognize two or more epitopes, have become increasingly of interest in diagnostic and therapeutic applications and can support novel mechanisms of action that are not available to monospecific antibodies. However, their generation presents challenges. Promiscuous pairing of heavy and light chains of two antibodies expressed in one cell can result in the production of 10 different molecules, with only one being bispecific and the remaining pairings resulting in non-functional or monospecific molecules.
Various strategies have been developed as an attempt to overcome this problem and encourage the correct assembly of the desired multispecific antibody. Such strategies for encouraging heterodimerization of two different heavy chains include techniques such as ‘knobs-into-hole’ (Ridgway 1990). Strategies for circumventing light chain mispairing include the use of a common light chain (Merchant 1998), domain swapping (Schaefer 2011), replacement of a native disulfide bond with an inter-chain one (Mazor 2015).
An example of a bispecific antibody format that incorporates some of these modifications to improve efficient production of these molecules is a “DuetMab” described in WO 2013/096291. DuetMab antibody molecules use knobs-into-holes technology for heterodimerization of 2 distinct heavy chains and increases the efficacy of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond.
While the above strategies have come some way to reduce chain mispairing, there remains a need to further improve pairing of polypeptide chains in multispecific antibodies and facilitate their efficient production. The present disclosure has been devised in light of the above considerations.
Amino acid residues at the interface between a lambda LC and HC where charge pairs could be introduced were identified, demonstrating that the introduction of these lambda charge pairs could advantageously improve chain pairing beyond what was achieved in a previous antibody format.
Accordingly, in one aspect provided herein is a multispecific antibody comprising:
In some aspects, lambda charge pair is located at position 117 in the CLλ and position 141 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, the lambda charge pair is selected from a. to e. of the above list. In some aspects, the lambda charge pair is selected from any one of a., b., and e. of the above list. In some aspects, the lambda charge pair is selected from a. and b. of the above list. In some aspects, the lambda charge pair is arginine at position 117 of the CLλ and aspartic acid at position 141 of the first CH1.
In some aspects, lambda charge pair is located at position 117 in the CLλ and position 185 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, lambda charge pair is located at position 119 in the CLλ and position 128 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, lambda charge pair is located at position 134 in the CLλ and position 128 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, lambda charge pair is located at position 134 in the CLλ and position 145 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, lambda charge pair is located at position 134 in the CLλ and position 183 in the first CH1. In some aspects, the lambda charge pair is the lambda charge pair is a lysine at position 134 of the CLλ, and an aspartic acid or a serine at position 183 of the first CH1.
In some aspects, lambda charge pair is located at position 136 in the CLλ and position 185 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
In some aspects, lambda charge pair is located at position 178 in the CLλ and position 173 in the first CH1. In some aspects, the lambda charge pair is selected from the following list:
As further described herein, the lambda charge pairs can be combined with other approaches for encouraging light chain pairing, for example in order to further increase the correct assembly of the desired multispecific antibody.
In some aspects, the multispecific antibody has the native inter-chain disulfide bond in one of the CH1-CL interfaces replaced with an engineered inter-chain disulfide bond. This was one of the approaches taken in the formation of the DuetMab format described in Mazer 2015. In some aspects:
In some aspects, the pair of cysteines engineered into the light chain and CH1 are located at position 122 of the light chain and position 126 of the CH1, and wherein the light chain comprises a non-cysteine residue at position 212 and the CH1 comprises a non-cysteine residue at position 220. In some aspects, the non-cysteine residues are valines.
In some aspects, the CLλ of the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 provides an exemplary wild type (native) CLλ, while SEQ ID NO: 2 provides an exemplary CLλ with the cysteine involved in the native inter-chain disulfide bond replaced with an engineered cysteine, for forming an engineered disulfide bond.
In some aspects, the first CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4 or SEQ ID NO: 5. SEQ ID NO: 4 provides an exemplary wild type (native) CH1, while SEQ ID NO: 5 provides an exemplary CH1 with the cysteine involved in the native inter-chain disulfide bond replaced with an engineered cysteine, for forming an engineered disulfide bond.
In some aspects, the second light chain comprises a constant light chain kappa region (CLκ). As described herein, the use of different light chains (lambda and kappa) is advantageous as it allows for methods such as light chain affinity chromatography to be used to selectively purify those multispecific antibodies containing the correct light chains. The inclusion of a kappa light chain in the multispecific antibody also allows for the inclusion of kappa charge pairs, which can encourage pairing of the second CH1:CLκ polypeptides.
In some aspects, the second antigen binding arm comprises a kappa charge pair located in the CLκ of the second light chain and in the second CH1, wherein the kappa charge pair of the second antigen binding arm comprises a positively charged amino acid residue selected from arginine, lysine or histidine located at one of the positions in the kappa charge pair of the second antigen binding arm and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine or threonine located at the other position in the kappa charge pair of the second antigen binding arm.
In some aspects, the negatively charged amino acid residue in the kappa charge pair of the second antigen binding arm is at position 133 of the CLκ, and the positively charged amino acid residue in the kappa charge pair is at position 183 of the second CH1. In some aspects, the negatively charged amino acid residue at position 133 of the CLκ is a glutamic acid, and wherein the positively charged amino acid residue at position 183 of the second CH1 is a lysine.
In some aspects, the CLκ of the second light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.
In some aspects, the second CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 1 or SEQ ID NO: 2.
In some aspects, the first antigen binding arm and/or second antigen binding arm comprises an Fc region. In some aspects, the first antigen binding arm comprises a first Fc region and the second antigen binding arm comprises a second Fc region. Various strategies can be used to encourage heterodimerization of the two heavy chains (i.e. heterodimerization of a first heavy chain containing the first CH1 and first Fc region, and a second heavy chain containing the second CH1 and second Fc region).
In some aspects, the first and second Fc regions comprise modifications to facilitate heterodimerization of the first and second Fc regions. In some aspects, these modifications are located in the CH3 of the Fc regions.
In some aspects, the modification in the CH3 of one of first and second Fc regions is a substitution of an amino acid residue with one having a larger side chain, thereby generating a protuberance (knob) on the surface of said CH3 domain, and the modification in the CH3 of the other Fc region is a substitution of an amino acid residue with one having a smaller side chain, thereby generating a cavity (hole) on the surface of said CH3 domain, optionally wherein the CH3 domain containing the protuberance (knob) is part of the first heavy chain polypeptide and the CH3 domain containing the cavity (hole) is part of the second heavy chain.
In some aspects, wherein the substitution to generate a knob is a substitution to tryptophan at position 366 and the substitution to generate a hole is a substitution comprising one or more of the following:
In some aspects, the CH3 domain containing the protuberance (knob) comprises a cysteine at position 354 and the CH3 domain containing the cavity (hole) comprises a cysteine at position 349.
In some aspects, the multispecific antibody comprise the lambda charge pairs in combination with any one or more of the engineered disulfides, kappa charge pairs and Fc modifications to facilitate heterodimerization described herein. For example, in some aspects, the multispecific antibody comprises the lambda charge pairs in combination with the engineered disulfides described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with the kappa charge pairs described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with the Fc modifications to facilitate heterodimerization described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with engineered disulfides and Fc modifications to facilitate heterodimerization described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with kappa charge pairs and Fc modifications to facilitate heterodimerization described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with engineered disulfides and Fc modifications to facilitate heterodimerization described herein. In some aspects, the multispecific antibody comprises the lambda charge pairs in combination with engineered disulfides, kappa charge pairs and Fc modifications to facilitate heterodimerization described herein.
Also provided herein is a method of producing the of producing the multispecific antibody described herein. In some aspects, the method comprises
In some aspects, the method comprises producing the multispecific antibody, the method comprising expressing the first, second and third light chain and the first, second and third CH1 in a host cell; wherein the first light chain pairs with the first CH1 so as to form the first binding arm, wherein the second light chain pairs with the second CH1 so as to form the second binding arm, wherein the third light chain pairs with the third CH1, and wherein the first binding arm pairs with the second and the third binding arms so as to form the multispecific antibody; and purifying the multispecific antibody from the host cell.
In some aspects, purifying the multispecific antibody comprises affinity chromatography. In some aspects, the purifying the multispecific antibody comprises light chain affinity chromatography.
In some aspects, less than 25%, less than 20%, less than 15%, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the light chains are mispaired following purification of the multispecific antibody. In some aspects, less than 10% of the light chains are mispaired. In some aspects, less than 5% of the light chains are mispaired. Suitable methods for determining the percentage of mispairing are described herein.
Also provided herein is one or more nucleic acid(s) encoding the first light chain and/or the first CH1 of the multispecific antibody described herein. In some aspects, the one or more nucleic acids further encode the second light chain and/or the second CH1. In some aspects, the one or more nucleic acid are part of a vector. Also provided herein is an isolated host cell comprising the nucleic acid(s) or vector.
Also provided herein are pharmaceutical compositions and therapeutic methods involving the pharmaceutical compositions or multispecific antibodies, as further described below.
Aspects and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:
Aspects and aspects of the present disclosure will now be discussed with reference to the accompanying figures. Further aspects and aspects will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Provided herein, as more fully discussed below, are multispecific antibodies (e.g. a bispecific antibody) comprising lambda charge pairs located at certain pairs of positions in the constant light chain lambda region (CLλ) and a heavy chain constant region 1 (CH1) in one binding arm of the multispecific antibody.
Methods are known for generating bispecific antibodies. Such methods, however, are often limited by a multitude of possible antibody formations which can include several combinations of incorrect pairings of heavy and light chains. Such mispairings can decrease production efficiency. The use of lambda charge variants as described herein overcome these limitations by preferentially causing the lambda light chain to pair with the correct CH1 in one binding arm, generating the preferred bispecific antibody assembly. In particular, these lambda charge variants can be combined with known approaches used to promote the correct pairing of heavy and light chains, such as Knobs into Holes (KiH), engineered disulfides and kappa charge pairing, as described in more detail below, to further improve the formation of the preferred bispecific antibody.
The term “antibody” or “antibody molecule” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The antibody may be human or humanized. In some aspects, the antibody is a monoclonal antibody molecule. Examples of antibodies are the immunoglobulin isotypes, such as immunoglobulin G (IgG), and their isotypic subclasses, such as IgG1, IgG2, IgG3 and IgG4, as well as fragments thereof.
An antibody is composed of two different types of polypeptide chain: one termed a heavy chain and the other terms a light chain. A natural monospecific antibody consists of two identical heavy chains and two identical light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. The disulfide bonds linking the light and heavy chains are sometimes termed “inter-chain” disulfide bonds, to distinguish them from the “intra-chain” disulfide bonds that are present within the individual heavy and light chain polypeptides.
Light chains in natural antibodies are either “lambda (λ)” or kappa “(κ)” light chains, which differ in terms of their amino acid sequence. Light chains are composed of a single constant light chain region (CL) and a single light chain variable region (VL). An example of a constant light chain lambda region (CLλ) amino acid sequence is provided as SEQ ID NO: 1 and an example of a constant light chain kappa region (CLκ) amino acid sequence is provided as SEQ ID NO: 3. Light chains used in the multispecific antibodies described herein may be chimeric light chains, e.g. contain a CLλ and a VLκ.
IgG heavy chains are composed of a heavy variable (VH) region and three heavy constant regions (CH1, CH2 and CH3), with an additional “hinge region” between CH1 and CH2. An example of an IgG1 CH1 region amino acid sequence is provided as SEQ ID NO:4. An example of an IgG1 CH2 amino acid sequence is provided as SEQ ID NO:6. An example of an IgG1 CH3 amino acid sequence is provided as SEQ ID NO: 7. An example of an heavy chain amino acid sequence comprising a CH1, hinge, CH2 and CH3 is provided as SEQ ID NO: 8.
Unless otherwise specified, amino acid residue positions in the constant domain, including the position of amino acid sequences, substitutions, deletions and insertions as described herein, are numbered according to EU numbering (Edelman, 2007).
The light chain associates with the VH and CH1 to form an “antigen binding arm” and the variable domains in the antigen binding arm interact to form the “antigen binding domain”.
An “antigen binding domain” describes the part of a molecule that binds to all or part of the target antigen and generally comprises six complementarity-determining regions (CDRs); three in the VH region: HCDR1, HCDR2 and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. The six CDRs together define the paratope of the antigen binding domain, which is the part of the antigen binding domain which binds to the target antigen. A monoclonal monospecific IgG antibody molecule contains two antigen binding domains, each of which are able to bind the same target (i.e. it is bivalent for a single target).
The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C term; and VL regions comprise the following structure: N term-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C term.
The present disclosure provides multispecific antibodies. Multispecific antibodies according to the present disclosure may be provided in isolated form, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides and/or serum components.
The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. The term “disulfide link” or “disulfide linked” as used herein, refers to the single covalent bond formed from the coupling of thiol groups, especially of cysteine residues. In some aspects, the covalent linkage between two cysteines is between the two sulfur atoms of each residue. However, depending on the environment, not all protein species may have a disulfide present at all times, for example, in the event of disulfide reduction. Thus, the term “disulfide link” or “disulfide linked” (whether native or engineered), in some aspects, also refers to the presence of two cysteine residues that are capable of forming a disulfide link, irrespective of whether or not they are actually linked at that individual point in time.
Multispecific antibodies of the present disclosure are capable of binding at least two epitopes, either on the same or different antigens, and comprise at least two antigen binding arms, referred to herein as a “first antigen binding arm” and a “second antigen binding arm”. According to the present disclosure, an “antigen binding arm” comprises a light chain, a VH and CH1 (i.e. at least one constant and one variable domain of each of the heavy and light chain), where the light chain is disulfide linked to the CH1. Each antigen binding arm may further comprise additional heavy chain regions, i.e. one or more of the hinge, CH2 and CH3. In some aspects, the antigen binding arm further comprises an Fc region (i.e. the remainder of the heavy chain comprising the hinge, CH2 and CH3). In some aspect, the multispecific antibody comprises complete heavy chains (i.e. a VH, CH1, hinge, CH2 and CH3).
The first and second antigen binding arms differ from each other in at least their light chain amino acid sequences and CH1 amino acid sequences (i.e. the first light chain and second light chain have different amino acid sequences, and the first CH1 and second CH1 have different amino acid sequences). In some aspects, the heavy chain of the first antigen binding arm is capable of forming a disulfide link to the heavy chain of the second antigen binding arm (e.g. via inter-chain disulfide bonds between cysteines present in the Fc domains).
Examples of multispecific antibodies include bispecific antibody, which are capable of binding to two epitopes, and a trispecific antibody, which are capable of binding to three epitopes, and so on. In some cases, the multispecific antibody is a bispecific antibody.
Multispecific antibody molecules may be provided in any suitable format. Suitable formats for a bispecific antibody molecule described herein, and methods for producing the same, are described in Kontermann, MAbs 2012, 4 (2): 182-197 and Kontermann and Brinkmann 2015, 20 (7): 838-847, both of which are herein incorporated by reference in their entirety. See in particular FIG. 2 of Kontermann MAbs 2012, 4 (2): 182-19. Particular examples of multispecific antibody formats include DuetMab, kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pairs and SEED-body. In certain aspects, the multispecific antibody is a DuetMab.
A particular exemplified format of asymmetrical IgG-like bispecific antibody molecules is referred to as “DuetMab”. DuetMab antibody molecules uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficacy of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Disclosure related to DuetMab can found e.g., in U.S. Pat. No. 9,527,927 and Mazor, 2015, which are herein incorporated by reference in their entirety, as is further described below.
The terms “charge pair(s)” and “charge mutation(s)” are used interchangeably throughout this specification and refer to a positively charged amino acid residue and a negatively charged amino acid residue, one of which is located in the a light chain region (e.g. constant light chain region) and the other in a heavy chain region (e.g. constant heavy chain region 1 (CH1)) of an antigen binding arm, located at positions intended to promote association of the light and heavy chains. By “lambda charge pair”, it is meant an introduced or substituted charge pair where a positively or negatively charged amino acid residue is located in a lambda light chain (e.g. CLλ) and a heavy chain constant region (e.g. CH1). By “kappa charge pair”, it is meant an introduced or substituted charge pair where positively or negatively charged amino acid residue in the light chain is located in a kappa light chain (e.g. CLκ) and a heavy chain constant region (e.g. CH1).
Without wishing to be bound by theory, it is believed that the oppositely charged amino acid residues in the charge pair increase the attraction of the heavy chain to the light chain in an antigen binding arm, thereby promoting formation of the antigen binding arm with the correct heavy and light chain.
At least one of the amino acid residues of the charge pair have been engineered into the antigen binding arm (i.e. at least one amino acid residue in the pair is not a wild-type amino acid residue). In some aspects, both amino acid residues in the charge pair are engineered into the antigen binding arm (i.e. both amino acid residues in the pair are not wild-type amino acid residues).
The amino acid residues of the charge pair are typically naturally occurring. Naturally occurring positively charged amino acid residues according to the present disclosure include arginine, lysine and histidine. Naturally occurring negatively charged amino acid residues according to the present disclosure include glutamic acid, serine, threonine and aspartic acid. Although serine and threonine are often described in the art as ‘uncharged’, they have an isoelectric point below 6 and therefore are partially negatively charged at neutral pH. For the purposes of the charge pairs disclosed herein, serine and threonine are examples of negatively charged amino acid residues (together with glutamic acid and aspartic acid). Hence, a charge pair may comprise a positively charged amino acid residue selected from arginine, lysine or histidine located at one of the positions in the charge pair and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine or threonine located at the other position in the charge pair. For example, the charge pair may comprise any one of the following pairs of amino acid residues:
In some aspects, the positively charged amino acid residue in the charge pair is located on the light chain and the negatively charged amino acid residue in the charge pair is located on the heavy chain. In other aspects, the negatively charged amino acid residue is located on the light chain and the positively charged amino acid residue in the charge pair is located on the heavy chain.
The multispecific antibodies described herein comprise a lambda charge pair in one of the antigen binding arms (also referred to herein as a “first antigen binding arm”). As exemplified herein, lambda charge pairs can be introduced at several positions to improve pairing of the correct light and heavy chains in the antigen binding arm.
In some aspects, the lambda charge pair comprises a positively or negatively charged amino acid residue at position 117, 119, 134, 136 or 178 of the constant light chain lambda region (CLλ). In some aspects, the lambda charge pair comprises a positively or negatively charged amino acid residue at position 141, 185, 128, 145, 183, 185, 173, or 187 of the CH1. As noted elsewhere, the numbering is according to EU numbering. Positions 117, 119, 134, 136, and 178 of the CLλ according to EU numbering corresponds to amino acid positions 10, 12, 27, 29, and 71 of SEQ ID NOs: 1 and 2. Positions 141, 185, 128, 145, 183, 185, 173, and 187 of the CH1 according to EU numbering corresponds to amino acid positions 24, 68, 11, 28, 66, 68, 56, and 70 of SEQ ID NOs: 4 and 5.
In some aspects, lambda charge pair located at one or more of the following pairs of positions:
In some aspects, the lambda charge pair is located at charge pair is located at position 117 in the CLλ and position 141 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is selected from any one of a. to f. of the above list. In some aspects, the lambda charge pair is selected from any one of a. to e. of the above list. In some aspects, the lambda charge pair is selected from any one of a., b., and e. of the above list. In some aspects, the lambda charge pair is a.
In some aspects, the lambda charge pair is located at charge pair is located at position 117 in the CLλ and position 185 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is located at charge pair is located at position 119 in the CLλ and position 128 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is located at charge pair is located at position 134 in the CLλ and position 128 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is located at charge pair is located at position 134 in the CLλ and position 145 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is located at charge pair is located at position 134 in the CLλ and position 183 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is a lysine at position 134 of the CLλ, and an aspartic acid or a serine at position 183 of the CH1. In the CH1 sequences provided as SEQ ID NO: 4 or SEQ ID NO: 5, EU position 183 is a serine and therefore it is not necessary to introduce a modification in the CH1 of SEQ ID NO: 4 or SEQ ID NO: 5 in order to produce a charge pair with a positively charged amino acid at position 134 of the CLλ.
In some aspects, the lambda charge pair is located at charge pair is located at position 136 in the CLλ and position 185 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the lambda charge pair is located at charge pair is located at position 178 in the CLλ and position 173 in the CH1. For example, the lambda charge pair can be selected from the following list:
In some aspects, the first antigen binding arm comprises more than one lambda charge pair. For example, the first antigen binding arm may comprise two, three, four, five, six, seven, eight or nine lambda charge pairs at positions (i) to (ix) described above.
In the multispecific antibodies described herein, one of the antigen binding arms (e.g. first antigen binding arm) comprises one of the lambda charge pairs described above, and another antigen binding arm (e.g. second antigen binding arm) comprises either a different lambda charge pair described above, or does not comprise a lambda charge pair.
For example, the first and second antigen binding arms in the multispecific antibody may both comprise lambda charge pairs described above, wherein the lambda charge pair in the first antigen binding arm is different to the lambda charge pair in the second antigen binding arm. That is, the lambda charge pair in the first antigen binding arm may be located at any one of the pairs of positions at (i) to (ix) described above and the lambda charge pair in the second antigen binding arm located at a different pair of positions within (i) to (ix) described above. For example, the first antigen binding arm may comprise a lambda charge pair at position 117 in the CLλ of the first antigen binding arm and position 141 in the first CH1 and the second antigen binding arm may comprise a different lambda charge pair at e.g. position 134 in the CLλ of the second antigen binding arm and position 145 in the second CH1.
In another example, the first antigen binding arm and second antigen binding arms in the multispecific antibody both comprise a lambda charge pair at the same position (i.e. at one of (i) to (ix) described above), but the positively and negatively charged amino acid residues are on different polypeptide chains in the respective antigen binding arms. That is, the first antigen binding arm may comprise a positively charged amino acid residue at one of the positions in the CLλ and a negatively charged amino acid residue in the first CH1, and the second antigen binding arm comprises a negatively charged amino acid residue at the same position of the CLλ and a positively charged amino acid residue at the same position in the second CH1, or vice versa. For example, the first antigen binding arm may comprise a lambda charge pair that is an arginine at position 117 of the CLλ of the first antigen binding arm and aspartic acid at position 141 of the first CH1, and the second antigen binding arm may comprise a lambda charge pair that is aspartic acid at position 117 in the CLλ of the second antigen binding arm and arginine at position 141 of the second CH1.
In further examples, the first antigen binding arm comprises one or more than one of the lambda charge pairs described above and the second antigen binding arm comprises a constant light chain kappa region (CLκ) (optionally with a kappa charge pair), as described in more detail below.
As demonstrated herein, multispecific antibodies containing lambda charge pairs exhibit improved correct light chain pairing when compared to multispecific antibodies lacking the lambda charge pair. That is, when producing the multispecific antibodies containing the lambda charge pair in the first antigen binding arm, the proportion of multispecific antibody containing the correct first light chain and first CH1 is increased compared to production of the equivalent multispecific antibody without the lambda charge pair.
As described in the examples, several methods are known that can be used to determine correct light chain paring. These include mass spectrometry-based approaches that can be used to establish association of the correct heavy/light chain. When the second binding arm comprises a kappa light chain, the ratio of kappa and lambda light chains in the assembled multispecific antibody can be determined using microfluidics-based electrophoresis as a readout of the correct light chain ratio.
Accordingly, in some aspects, the multispecific antibody containing the lambda charge pair exhibits improved correct light chain pairing when compared to an equivalent multispecific antibody that lacks the lambda charge pair. In some aspects, the multispecific antibody containing the lambda charge pair exhibits a correct light chain ratio greater than 90%, 95%, 96%, 97%, 98% or 99% (e.g. as determined using a microfluidics-based electrophoresis method), optionally after the multispecific antibody has been purified using light chain affinity purification.
Combination with Other Pairing Approaches
The lambda charge pairs described herein may be combined with other strategies for promoting heterodimerization in order to further increase the correct pairing of heavy and light chain polypeptides.
Non-limiting examples of strategies for promoting heterodimerization are described in more detail below and include using disulfide engineering at the CH1/CL interface, introducing additional charge pairs (e.g. kappa charge pairs) and Fc region modifications such as knobs-into-holes and allow fractionated purification strategies.
In some aspects, the multispecific antibodies contain engineered disulfides in addition to the lambda charge pairs. By “engineered disulfides” it is meant that a native inter-chain disulfide bond at the CH1-CL interface (e.g. at 220 of the CH1 and 212 of the LC) of one of the antigen binding arms has been replaced by an engineered (non-native) interchain disulfide, while the other antigen binding arm contains the native interchain disulfide bond at the CH1-CL interface. An engineered disulfide is typically formed by engineering cysteines into the CL of a light chain and the CH1 of the corresponding heavy chain and replacing the cysteines that normally form the interchain disulfide. Disclosure related to the introduction of engineered disulfide into multispecific antibodies for the purpose of promoting heterodimerization can found e.g., in U.S. Pat. No. 9,527,927 and Mazor, 2015, which are herein incorporated by reference in their entirety.
Thus, in some aspects:
In some aspects, the pair of cysteines engineered into the light chain and CH1 are located at position 122 of the light chain and position 126 of the CH1, and wherein the same light chain comprises a non-cysteine residue at position 212 and the same CH1 comprises a non-cysteine residue at position 220. In some aspects, the non-cysteine residues are valines.
An exemplary amino acid sequence of a CLλ comprising an engineered cysteine is provided as SEQ ID NO: 2 and an exemplary amino acid sequence of the CH1 comprising the corresponding engineered cysteine to form the engineered disulfide is provided as SEQ ID NO: 5.
In the multispecific antibodies exemplified herein, the engineered disulfide is present on the “first” antigen binding arm containing the lambda charge pairs and the native disulfide is present on the “second” antigen binding arm that does not contain the lambda charge pairs. However, the opposite arrangement is also specifically contemplated, i.e. where the native disulfide is present on the first antigen binding arm and the engineered disulfide is present on the second antigen binding arm.
In some aspects, the pair of cysteines engineered into a constant light chain kappa region (CLκ) and CH1 are located at position 121 of the CLκ and position 126 of the CH1, and wherein the same CLκ comprises a non-cysteine residue at position 214 and the same CH1 comprises a non-cysteine residue at position 220. In some aspects, the non-cysteine residues are valines.
In some aspects, the second antigen binding arm comprises a constant light chain kappa region (CLκ). That is, one of the antigen binding arms contains an CLλ and a different antigen binding arm comprises an CLκ in the multispecific antibody. As described herein, techniques such as light chain affinity chromatography that utilizes affinity resins specific for either CLκ or CLλ can be used to selectively purify antibodies based on their light chain. Examples of such affinity resins include the LambdaFabSelect and KappaSelect resins available from GE Healthcare. Such methods can be used to selectively purify multispecific antibodies containing both CLκ and CLλ and can therefore be used to improve production of bispecific antibodies in this format.
An example of an CLκ amino acid sequence is provided as SEQ ID NO: 3.
In some aspects, the second antigen binding arm comprises a kappa charge pair. As described above, kappa charge pairs refer to a positively charged amino acid residue and a negatively charged amino acid residue, one of which is located in the kappa light chain (e.g. CLκ) and the other in the heavy chain (e.g. CH1) of an antigen binding arm, located at positions intended to promote association of the light chain and CH1 of the second antigen binding arm.
In some aspects, the second antigen binding arm comprises a kappa charge pair located at position 133 in the CLκ and position 183 in the second CH1. In some aspects, the negatively charged amino acid residue in the kappa charge pair is at position 133 of the CLκ and the positively charged amino acid residue in the kappa charge pair 183 of the second CH1. In other aspects, the positively charged amino acid residue in the kappa charge pair is at position 133 of the CLκ and the negatively charged amino acid residue in the kappa charge pair 183 of the second CH1. In some aspects, the negatively charged amino acid residue (e.g. at position 133 of the CLκ) is a glutamic acid, and wherein the positively charged amino acid residue (e.g. at position 183 of the second CH1) is a lysine. As noted elsewhere, this numbering is according to EU numbering.
Position 133 of the CLκ according to EU numbering corresponds to amino acid position 26 of SEQ ID NO: 3. Position 183 of the CH1 according to EU numbering corresponds to amino acid 66 of SEQ ID NOs: 4 and 5.
In certain aspects, the multispecific antibody comprises a first antigen binding arm with a lambda charge pair as described above and a second antigen binding arm with a kappa charge pair as described above, wherein the multispecific antibody comprises engineered disulfides.
For example, in one aspect, the first antigen binding arm comprises a lambda charge pair (e.g. at position 117 in the CLλ and position 141 in the first CH1) and the disulfide link between the first light chain and first CH1 is formed between a pair of cysteines engineered into the CLλ and first CH1; and the second antigen binding arm comprises a kappa charge pair (e.g. at position 133 in the CLκ and position 183 in the second CH1) and the disulfide link between the second light chain and second CH1 is formed between a pair of native cysteines in the CLκ of the second light chain and second CH1.
In another aspect, the first antigen binding arm comprises a lambda charge pair (e.g. at position 117 in the CLλ and position 141 in the first CH1) and the disulfide link between the first light chain and first CH1 is formed between a pair of native cysteines in CLλ of the first light chain and first CH1; and the second antigen binding arm comprises a kappa charge pair (e.g. at position 133 in the CLκ and position 183 in the second CH1) and the disulfide link between the second light chain and second CH1 is formed between a pair of cysteines engineered in the CLκ of the second light chain and second CH1.
As noted above, in some aspects the first and second antigen binding arms further comprise a first and second Fc region (i.e. further comprising the CH2 and CH3 regions of a heavy chain).
In some aspects, the antibody molecules comprise one or more modifications in one or more of the CH1, CH2 and CH3 domains that promotes formation of a heterodimeric antibody molecule by facilitating formation of the first and second Fc regions. This may involve a Knobs into Holes (KiH) strategy based on single amino acid substitutions in the CH3 domains that promote heavy chain heterodimerization as described in Ridgway, 1996. The knob variant heavy chain CH3 has a small amino acid has been replaced with a larger one, thereby generating a protuberance (knob) on the surface of said CH3 domain, and the hole variant has a large amino acid has replaced with a smaller one thereby generating a cavity (hole) on the surface of said CH3 domain. Additional modifications may also be introduced to stabilize the association between the heavy chains.
CH3 modifications to enhance heterodimerization include, for example, “hole” mutations Y407V/T366S/L368A on one Fc region and “knob” mutation T366W on the other Fc region. These may further include stabilizing cystine mutations Y349C (e.g. on the Fc region with the “hole” mutation) and stabilizing S354C mutation on the other Fc region (e.g. on the Fc region with the “knob” mutation. Exemplary amino acid sequences of a CH3 domain engineered to contain a “hole” mutation are provided as SEQ ID NOs: 9 and 10 Exemplary amino acid sequences of a CH3 domain engineered to contain a “knob” mutation are provided as SEQ ID NOs: 11 and 12.
Accordingly, in one aspect, the substitution to generate a knob is a substitution to tryptophan at position 366 and the substitution to generate a hole is one or more of the following:
In the multispecific antibodies exemplified herein, the “knob” is present on the “first” antigen binding arm containing the lambda charge pairs and the “hole” is present on the “second” antigen binding arm that does not contain the lambda charge pairs. However, the opposite arrangement is also specifically contemplated, i.e. where the “hole” is present on the CH3 of the first antigen binding arm and the “knob” is present on the CH3 of the second antigen binding arm.
For example, the one Fc region may include a modification to allow fractionated elution by protein A chromatography. Briefly, one of the Fc regions may comprise a modification that ablates binding to protein A (termed Fc*), allowing for selective purification of the heterodimeric FcFc* bispecific product. Examples of suitable modifications for generating an Fc* region include substitution of H435 with arginine and Y436 with phenylalanine.
In some aspects, the multispecific antibody comprises:
In some aspects, the multispecific antibody comprises:
In some aspects, the multispecific antibody comprises:
In some aspects, the multispecific antibody comprises:
Non-limiting examples of bispecific antibodies comprising a lambda charge pair, a kappa charge pair, engineered disulfides and modification to facilitate heterodimerization of the first and second Fc regions are provided in the examples.
Other Fc modifications contemplated herein are those that reduce or abrogate binding of the antibody molecule to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII and/or to complement. Such mutations reduce or abrogate Fc effector functions. Mutations for reduce or abrogate binding of antibody molecule to one or more Fcγ receptors and complement are known and include the “triple mutation” or “TM” of L234F/L235E/P331S (according to European Union numbering convention) described for example in Organesyan et al., Acta Crystallogr D Biol Crystallogr 64 (6): 700-704, 2008. In some aspects, the CH2 domain of either or both immunoglobulin heavy chain constant domains comprises the following substitutions: E233P/L234V/L235A/G236del/S267K. This combination of mutations may be referred to herein as the “Fc effector null mutation”.
Other suitable Fc region amino acid substitutions or modifications are known in the art and include, for example, the triple substitution methionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU index as in Rabat (M252Y/S254T/T256E; referred to as “YTE” or “YTE mutation”) (see, e.g., U.S. Pat. No. 7,658,921; U.S. Patent Application Publication 2014/0302058; and Yu et al., Antimicrob. Agents Chemother., 61 (1): e01020-16 (2017), each of which is herein incorporated by reference in its entirety). This combination of mutations may extend the half-life of the antibody.
The triple mutation, Fc effector null mutation and YTE mutation, when present, may be present in one or both heavy chain constant domains. Typically, if included, they are included in both heavy chain constant domains.
In some aspects, one of the antigen binding arms is capable of binding CD3.
CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3 associates with the T-cell receptor and the ζ chain to generate an activation signal in T lymphocytes. Bispecific antibodies that target CD3 and a target cell antigen have been used to force a temporary interaction between the target cell and T cell, causing cross-linking, T-cell activation, and subsequent antigen-dependent T cell killing of the target cell.
As described herein, a first antigen binding arm comprises a first light chain capable of forming a disulfide link to a first CH1, the first light chain comprising a constant light chain lambda region (CLλ). Also as described herein, a second antigen binding arm comprises a second light chain capable of forming a disulfide link to a second CH1. In some aspects, the second light chain comprises a constant light chain kappa region (CLκ).
In some aspects, the CLλ of the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the CLλ of the first light chain comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.
In some aspects, the CLκ of the second light chain (where present) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. In some aspects, the CLκ of the second light chain (where present) comprises an amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.
In some aspects, the first or second CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4 or SEQ ID NO: 5. In some aspects, the CH1 comprises an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.
The 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications may be in addition to the modifications described above to introduce the charge pairs, engineered disulfides and/or Fc region modifications described above. For example, compared to the wild type CLλ set forth in SEQ ID NO: 1, the CLλ used in the multispecific antibody may contain a lambda charge pair mutation, an engineered disulfide (e.g. S122C and C212V) and 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 further amino acid modifications. As another example, compared to the wild type CH1 provided in SEQ ID NO: 4, the first CH1 used in the multispecific antibody may contain a lambda charge mutation, an engineered disulfide (e.g. F126C, C220V) and 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 further amino acid modifications.
An amino acid modification may be an insertion, a substitution, or a deletion. In some aspects, the amino acid modification is a substitution of an amino acid residue to any other naturally occurring or non-naturally occurring amino acid residue.
Naturally occurring residues may be divided into classes based on common side chain properties:
As described above, serine(S) and threonine (T) have an isoelectric point below 6 and are partially negatively charged at neutral pH, hence they are classed here as ‘polar, partially negatively charged’.
The amino acid substitution may be a conservative amino acid substitution. Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. For example, a conservative amino acid substitution may be a substitution of the acidic amino acid glutamic acid (E) for the acidic amino acid aspartic acid (D).
Also provided herein is one or more nucleic acid(s) encoding the multispecific antibody described herein. In some aspects, the nucleic acid(s) is/are purified or isolated, e.g. from other nucleic acid, or naturally-occurring biological material. The skilled person would have no difficulty in preparing such nucleic acid molecules using methods well-known in the art.
In some aspects, the one or more nucleic acids encode a light chain as described herein and/or a CH1 as described herein. The one or more nucleic acid(s) encoding the first or second CH1 may further encode other heavy chain domains, e.g. the hinge, CH2 and CH3, and may encode a complete heavy chain.
The present disclosure also provides one or more vector(s) comprising nucleic acid(s) encoding a multispecific antibody described herein. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. In some aspects, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.
The multispecific antibody may be produced from a light chain vector and a heavy chain vector. A light chain vector may contain the nucleic acid encoding the first light chain and the nucleic acid encoding the second light chain, which may be present on the vector as separate cassettes (e.g. each operably connected to a different promoter). Similarly, a heavy chain vector may contain may be used to encode both the first CH1 (and first Fc region, if present) and second CH1 (and second Fc region, if present), which may be present on the vector as separate cassettes. Alternatively, separate vectors may be used to encode each of the first light chain, second light chain, first CH1 (and first Fc region, if present) and second CH1 (and second Fc region, if present).
A nucleic acid molecule or vector as described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid or vectors into host cells are well established in the art and any suitable technique may be employed. A range of host cells suitable for the production of recombinant antibody molecules are known in the art, and include bacterial, yeast, insect or mammalian host cells. In some aspects, the host cell is a mammalian cell, such as a CHO, NS0, or HEK cell, for example a HEK293 cell. In some aspects, the host cell is a CHO cell.
Also provided herein is a method of producing the multispecific antibody described herein. In some aspects, the method comprises a) expressing the first and second light chain and the first and second CH1 in a host cell; b) allowing the first light chain to pair with the first CH1 so as to form the first binding arm, and allowing the second light chain to pair with the second CH1 so as to form the second binding arm, and allowing the first binding arm to pair with the second binding arm so as to form the multispecific antibody; and c) purifying the multispecific antibody from the host cell.
Expressing the first and second light chain and first and second CH1 in a host cell may comprise introducing nucleic acids or vectors into host cells (e.g. CHO cells) using suitable techniques as described above. The host cell may then be cultured using suitable techniques, such that the light chain and heavy chain polypeptides pair and form the first and second binding arms. During normal bispecific antibody development, the various light chains and heavy chain polypeptides associate with each other (e.g. through inter-chain disulfide bonds formed between native cysteines, and/or through cysteines engineered into the bispecific antibodies as described herein) and the heavy chains associate with each other (e.g. through inter-chain disulfide bonds formed between cysteines in the two Fc domains). As described herein, the presence of the lambda charge pairs encourages the correct heavy chain/light chain pair to form in the bispecific antibody.
Techniques for the purification of recombinant antibody molecules are well-known in the art and include, for example high performance liquid chromatography, fast protein liquid chromatography, ion exchange chromatography, and affinity chromatography, e.g. using Protein A or Protein L or by binding to an affinity tag. In some aspects, purification is carried out using affinity chromatography (e.g. Protein A affinity chromatography). In some aspects, purification further comprises (e.g. in addition to Protein A chromatography) light chain affinity chromatography. As described herein, light chain affinity chromatography can be used to selectively purify multispecific antibodies containing both CLκ and CLλ and can therefore be used to improve production of bispecific antibodies in this format.
In some aspects, less than 25%, less than 20%, less than 15%, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the light chains in the multispecific antibodies are mispaired (i.e. paired with a CH1 from a different antigen binding arm) following purification (e.g. by protein A affinity chromatography, or following protein A affinity chromatography and light chain affinity chromatography). Methods for determining the correct light chain pairing are known in the art and include mass spectrometry analysis and microfluidics-based electrophoresis, as described in more detail herein. In some cases the method comprises measuring the correct light chain pairing.
The method may also comprise formulating the antibody molecule into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below.
The multispecific antibodies described herein may thus be useful for therapeutic applications, such as in the treatment of cancer.
An multispecific antibody as described herein may be used in a method of treatment of the human or animal body. Related aspects of the disclosure provide;
Treatment may be any treatment or therapy in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, ameliorating, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of an individual or patient beyond that expected in the absence of treatment.
Treatment as a prophylactic measure (i.e. prophylaxis) is also included. For example, an individual susceptible to or at risk of the occurrence or re-occurrence of a disease such as cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of the disease in the individual.
A method of treatment as described may be comprise administering at least one further treatment to the individual in addition to the multispecific antibody. The multispecific antibody described herein may thus be administered to an individual alone or in combination with one or more other treatments. Where the multispecific antibody is administered to the individual in combination with another treatment, the additional treatment may be administered to the individual concurrently with, sequentially to, or separately from the administration of the multispecific antibody. Where the additional treatment is administered concurrently with the multispecific antibody, the multispecific antibody and additional treatment may be administered to the individual as a combined preparation. For example, the additional therapy may be a known therapy or therapeutic agent for the disease to be treated.
Whilst an multispecific antibody may be administered alone, multispecific antibodies will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the multispecific antibody. Another aspect of the disclosure therefore provides a pharmaceutical composition comprising an multispecific antibody as described herein. A method comprising formulating a multispecific antibody into a pharmaceutical composition is also provided.
Pharmaceutical compositions may comprise, in addition to the multispecific antibody, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below.
Administration may be in a “therapeutically effective amount”, this being sufficient to show benefit to an individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and may depend on the severity of the symptoms and/or progression of a disease being treated.
The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilized for realizing the disclosure in diverse forms thereof.
While the disclosure has been described in conjunction with the exemplary aspects described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary aspects of the disclosure set forth above are considered to be illustrative and not limiting. Various changes to the described aspects may be made without departing from the spirit and scope of the disclosure.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another aspect. The term “about” in relation to a numerical value is optional and means for example +/−10%.
To improve correct chain pairing beyond what alternative disulfides can achieve in DuetMab setting (see WO 2013/096291, incorporated herein by reference), charge pairs were designed using amino acids that participate in lambda light chain (LC)-heavy chain (HC) interface. Previous strategies for improving HC LC chain pairing have engineered oppositely charged amino acid residues at the interface between a kappa LC and CH1. It was recognized that charge pairs engineered into the kappa LC-HC interface are unlikely to work in the same way when engineered into equivalent positions in the lambda/CH1 interface. For example the presence of Y178 in the lambda LC is expected to disrupt charge pairs engineered into V134 of the lambda LC (equivalent to V133 of the kappa LC) and S138 of CH1 (see
The following positions were evaluated as lambda light chain amino acids participating in interface formation with CH1 domain: T117, F119, S122, E124, E125, K130, T132, V134, L136, S138, D139, E161, T163, S166, Q168, A174, S176, Y178, S180, in connection with the following heavy chain CH1 domain amino acids participating in interface formation with lambda light chain CL domain: S124, F126, L128, A129, S131, S132, K133, S134, A141, G143, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S181, S183, V185, T187, V211, K213.
These amino acids were explored pairwise or alone, one pair at a time or in combinations, with alternative interchain disulfides or keeping disulfides native. Introduction of positively or partially positively charged amino acid means substituting existing amino acids at that position with lysine and arginine and in some cases with asparagine or glutamine or histidine. Introduction of negatively or partially negatively charged amino acid means substituting existing amino acids at that position with aspartic acid, glutamic acid, serine, threonine and in some cases with asparagine or glutamine. Addition of histidine residue at some of these positions will allow the introduction of a pH dependent CH1-CL interaction.
Nine sets of pair combinations in the lambda LC-HC interface, meeting the criteria mentioned above, are provided in Table 1 as a non-exhaustive list of examples and were tested for improved pairing in this specification.
Table 1. All presented here mutations are specific for lambda light chain containing molecules and expected to perform in wild type as well as V12 formats (see below). In addition, opposite charge pairs [i.e., V134 (D,E,S,T)-L128 (R,K,H)] are also expected to provide preferential pairing. Net no charge side chain containing amino acids like asparagine and glutamine can be used for substitutions for either bearing positive or negative partial charge as they have been found to participate in formation of hydrogen bonds with both positively and negatively charged amino acids as well as to each other.
The materials and methods set forth herein were used to perform the experiments described in subsequent examples. All reagents were from Thermo Fisher Scientific, Waltham, MA, unless stated otherwise. As noted elsewhere, the terms “charge pair(s)” and “charge mutation(s)” are used interchangeably throughout this specification and the amino acid numbering is based on EU numbering system unless specified otherwise.
Construction of pDuet-Heavy and pDuet-Light Mammalian Expression Vectors for DuetMab with Charge Pairs
For construction of DuetMab antibodies with charge pair mutations in heavy chain-light chain interface, the pDuet-Heavy and pDuet-Light plasmids described in (WO 2013/096291 and in Mazor et. al mAbs 2015) were used as backbone vectors. Briefly, the pDuet-Heavy vector contained two human gamma1 heavy chain (HC) cassettes to support HC heterodimerization, where the former heavy chain carried the “Hole” set of mutations (T366S/L368A/Y407V) and a stabilizing mutation (Y349C) in CH3 domain, while the latter carried the complement “Knob” mutation (T366W) and a stabilizing mutation (S354C) in CH3, although the order of the cassettes could readily be reversed. The pDuet-Light vector contained two human light chain (LC) cassettes, where the former light chain carried a kappa constant domain (CK), while the latter carried a lambda constant domain (CA). The pDuet-Heavy and pDuet-Light vectors also contained the mutations to remove the native interchain disulfide bond in CH1/CA and provide the alternative disulfide bond which is denoted as “V12 DS” or “V12” in this specification, where the mutations F126C/C220V were introduced in the CH1 domain of the “Knob” heavy chain, and mutations S122C/C212V were introduced in the lambda constant domain. The amino acid sequences of the constant domains in the exemplified DuetMab antibody backbones (prior to the introduction of charge mutations) is provided as follows:
The mutations of the “Knob-and-Hole” set and the stabilizing/alternative disulfide bonds utilized herein were provided merely as an example. One skilled in the art can use any other combinations of mutations for “Knob-and-Hole” technique and/or stabilizing/alternative disulfide bonds known in this field to support HC heterodimerization.
For construction of the pDuet-Heavy vector with charge mutations, the “Hole” heavy chain was cloned into the pDuet-Heavy vector by a synthesized DNA fragment of VH-CH1-CH2-CH3 domains containing the above-mentioned mutations for “Hole” heavy chain using restriction cloning technique by BssHII/HindIII. Optionally, the “Hole” heavy chain contained the charge mutation S183K in CH1 domain. The “Knob” heavy chain was cloned into the vector by a synthesized DNA fragment of VH-CH1-CH2-CH3 domains containing the above-mentioned mutations for “Knob” heavy chain using restriction cloning technique by BsrGI/EcoRI. Optionally, the “Knob” heavy chain contained one of the charge mutations in CH1 domain: L128D, L128E, L128S, L128T, A141D, A141E, A141S, A141T, L145D, L145E, L145S, L145T, S183D, V185D, V185E, V185S, V185T, V173D, V173E, V173S, and V173T.
For construction of the pDuet-Light with charge mutations, the kappa light chain was cloned into the pDuet-Light vector by a synthesized DNA fragment of VL-Cκ domains using restriction cloning technique by BssHII/NheI. Optionally, the constant kappa (Cκ) domain contained the charge mutation V133E. The lambda light chain was cloned into the pDuet-Light vector by a synthesized DNA fragment of VL-Cλ domains containing the above-mentioned S122C/C212V mutations for lambda light chain using restriction cloning technique by BsrGI/EcoRI. Optionally, the constant lambda (Cλ) domain contained one of the charge mutations: V117R, V117K, F119R, F119K, V134R, V134K, L136R, L136K, Y178R, and Y178K. The light chain variable domain (VL) could be either variable kappa domain (Vκ) or variable lambda domain (Vλ).
All constructs were transiently expressed in CHO cells in suspension using PEI-MAX (Polysciences, Inc., Warrington, PA) as a transfection reagent and grown in an in-house made CHO medium. The vectors containing the following combinations of charge pairs were used for the expression of the antibodies in these studies. A schematic of the constructed DuetMabs containing charge pairs is provided in
The culture medium was collected 7 to 13 days after transfection and filtered through a 0.22 μm sterile filter. Antibody concentration in culture supernatants was measured by an Octet384 instrument using protein A sensors (Sartorius, Göttingen, Germany) according to the manufacturer's protocol. Antibodies were purified by either protein A magnetic bead affinity purification (Genscript, Piscataway, NJ) or standard protein A affinity chromatography (Cytiva, Marlborough, MA), followed by light chain affinity chromatography if necessary, in accordance with the manufacturer's protocol, and were subsequently buffer exchanged in PBS (pH 7.2). The purity and oligomeric state of purified molecules was determined by microfluidics-based electrophoresis and analytical size exclusion chromatography (see methods below). Protein aggregates were removed by preparative SEC. The concentrations of the purified antibodies were determined by reading the absorbance at 280 nm using theoretically determined extinction coefficients.
Analytical SEC-HPLC (Agilent 1260 Infinity HPLC system) was performed using a TSK-gel G3000SWxL column (Tosoh Biosciences, King of Prussia, PA) to determine the oligomeric state of purified molecules. Preparative SEC-HPLC was carried out using a Superdex 200 column (Cytiva) to remove protein aggregates.
Microfluidics-based electrophoresis was performed using Bioanalyzer in accordance with the manufacturer's protocol (Agilent, Santa Clara, CA), in order to assess the ratio of kappa and lambda light chains of an antibody, based on which the percentage of correct light chain ratio was calculated.
Binding kinetics were measured by biolayer interferometry on an Octet384 instrument. Streptavidin (SA) biosensors were loaded with biotinylated protein antigens (ACRO Biosystems, Newark, DE) in PBS pH 7.2, 1 mg/ml BSA, 0.05% (v/v) TWEEN (Kinetic buffer). The loaded biosensors were washed in the same buffer before carrying out association and dissociation measurements with various antibodies for the indicated times. Kinetic parameters (Kon and Koff) and affinities (KD) were calculated from a non-linear fit of the data using the Octet384 software v.12.2.1.24.
Protein test samples were diluted to 1 mg/mL in PBS (pH 7.2) and split into 3 equal aliquots to serve as control, heat, and photo stress samples. Control samples were incubated at 4° C. for 14 days, heat stress samples were incubated at 45° C. for 14 days, and photo stress samples were incubated in glass vials in an ICH compliant photostability chamber exposed to 3000 lux cool white light for 7 days at 25° C. Samples were then analyzed by HP-SEC to determine levels of aggregate, monomer, and fragment.
Samples were prepared by combining 20 μL of protein sample at 1 mg/mL in PBS (pH 7.2) with 5 μL of SYPRO Orange dye diluted to 40× in PBS (pH 7.2) in a 96-well PCR plate in duplicate. The plate was sealed, and measurements performed in a QuantStudio 7 Flex Real-Time PCR System. Samples were subjected to an initial equilibration step at 25° C. for 2 minutes, followed by a temperature ramp to 99° C. at 0.05° C./see increments. The fluorescence emission was monitored using the FAM filter set. The Tm value for each sample was calculated in the Protein Thermal Shift™ software using the Boltzmann method.
Subunit LC/MS analysis was performed to characterize the mis-paired species. 50 μg of sample was dried and further reconstituted in 50 μL of 100 mM sodium phosphate buffer, pH 7.0. Digestion was performed by adding 60 units of FabALACTICA enzyme (IgdE) (Genovis AB, Lund, Sweden) to each sample and incubating at 37° C. for 16-18 hours. Waters ACQUITY UPLC system (Waters, Milford, MA) coupled with Waters Xevo G2-XS QTof mass spectrometer were used for subunits separation and mass determination. Two μg of digested subunits were injected in Waters BioResolve RP mAb polyphenyl column (2.1×150 mm, 2.7 mm, 450 Å) for separation. Mobile phase A contained 0.1% formic acid (FA), 0.01% trifluoroacetic acid (TFA) in water, and mobile phase B contained 0.1% FA, 0.01% TFA in water in ACN. A gradient of 25% B to 45% B was performed for 40 minutes at a flow rate of 0.2 mL/min. Column temperature was set at 75° C. The UV profile of eluted subunits were acquired at a wavelength of 280 nm.
DSC experiments were carried out using a MICROCAL VP-DSC scanning microcalorimeter (Malvern, Northampton, MA). Prior to DSC analysis, all samples were diluted to ˜0.6 mg/mL in phosphate buffer saline (PBS, pH 7.2). Exact concentrations were determined from duplicate measurements using a UV-VIS spectrophotometer (NanoDrop 2000C). 400 μL of each sample and corresponding buffer (PBS, pH 7.2) were loaded into a 96-well plate and stored at 10° C. in the autosampler chamber until analysis. All DSC measurements used a temperature window from 20° C. to 100° C. at a scan rate of 60° C./hr. Prior to sample measurement, baseline measurements (buffer-versus-buffer) were obtained for subtraction from the sample measurement. Data analysis, baseline correction, and deconvolution were carried out using the Origin™ DSC software provided by Microcal. Baseline correction was performed using the Linear Connect function within the software. Deconvolution analysis was performed using a non-two-state model and best fits were obtained using 1 and 200-iteration cycles until the chi-square value was minimized. The interpretation of the DSC deconvolution results was based on the fact that the different domains in the antibody formats unfold independently. The Tonset value is defined as the temperature at which the thermogram begins to significantly increase from the baseline. The Tm value is defined as the temperature value corresponding to each peak maximum on the thermogram or the deconvoluted thermogram.
Cell viabilities were determined using CellTiter-Glo™ Luminescent Cell Viability Assay (Promega). This assay quantifies the ATP present, which signals the presence of metabolically active cells. Luminescence, produced by the luciferase-catalyzed reaction of luciferin and ATP, was measured using a luminescent plate reader. In brief, target cells (NCI H358) were seeded in 96-well plates at a density of ˜ 1×104 cells/well in RPMI 1640 media supplemented with 0.1% BSA, and 0.2 ng/ml human recombinant EGF. Antibodies at various concentrations were added to triplicate samples, and the cells were incubated for 72 hours at 37° C. and 5% CO2 in a humidified incubator. After treatment, the cells were exposed to CellTiter-Glo® reagent (Promega) for ˜15 min and OD409 was measured using an EnVision 2104 Multilabel plate reader (PerkinElmer). Cell viability was determined by measuring the ATP level relative to a no-antibody control.
Table 3 and
Table 4 summarizes the expression (Table 4A) and biochemical profiles (Table 4B) of Antigen 1/Antigen 2 DuetMabs carrying the selected charge pair variants #1, #2, #33, #34, #35, #36, and #41 produced in large scale of cell culture (100 mL). The biochemical profiles of the selected DuetMabs were consistent despite of the production scale. For additional analysis, the DuetMabs were further purified by light chain affinity chromatography to remove mispaired byproducts, and aggregates were removed by preparative SEC.
Table 6 summarizes the thermal stabilities of Antigen 1/Antigen 2 DuetMabs carrying the selected charge pair variants by differential scanning fluorimetry (DSF) and their accelerated stability profiles. NIP228 served as an IgG1 control. The Antigen 1/Antigen 2 DuetMab variants showed no flags for aggregation or fragmentation after heat stress. HP-SEC retention times of the Antigen 1/Antigen 2 DuetMab variants are consistent with that of NIP228 IgG1 control (ΔRT from NIP228<0.2 m). DSF values showed no significant difference among the charge pair variants and were consistent with that of NIP228 IgG1 control.
X-ray crystallography was carried out in order to further investigate the lambda charge variants at the light chain: CH1 interface.
The coding sequences for (i) the variable domain of a light chain from an anti-Antigen 2 antibody and the constant domain of human lambda light chain containing T117R, S122C, and C212V mutations, and (ii) the variable domain of an anti-Antigen 2 antibody heavy chain and CH1 domain containing A141D or A141E as well as F126C and C220V mutations were ordered as synthetic DNA gBlocks from Integrated DNA Technologies (Coralville, IA). The coding sequence of light chain was flanked by N-terminal BssHII and C-terminal NheI restriction sites, and the heavy chain was flanked by N-terminal BsrGI and C-terminal EcoRI restriction sites to facilitate cloning. The gBlocks were digested and inserted into a mammalian expression vector (pOE; AstraZeneca, Gaithersburg, MD). One Shot Top10 chemically competent Escherichia coli cells (Invitrogen, Carlsbad, CA) were used as the host for gene cloning.
Both Fabs were transiently expressed in a suspension of human embryonic kidney (HEK) 293 cells, using 293fectin Transfection Reagent (Life Technologies, Carlsbad, CA) and standard protocols. Cells were grown in FreeStyle 293-F Expression Medium (Life Technologies) for 10 days and fed with a proprietary cell feed solution (AstraZeneca), after which the suspension was spun down and the supernatant filtered through a 0.2 μM filter. The Fab was purified from the supernatant using a 5 ml CaptureSelect CH1-XL column (Thermo Fisher Scientific, Waltham, MA), dialyzed against 25 mM Hepes pH 7 and further polished with a 5 ml HiTrap SP HP cation exchange column (Cytiva, Marlborough, MA) in a NaCl gradient in order to improve the homogeneity of the sample.
The Fabs were individually run on a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with 25 mM HEPES, pH 7.5 and 100 mM NaCl to ensure homogeneity of the samples before setting up crystallization screens. Initial crystallization trials for both proteins were carried out by the sitting-drop vapor-diffusion method at 20° C. The crystallization drops were dispensed in 96-well crystallization plates (Intelli-Plate 102-0001-20; Art Robbins Instruments, Sunnyvale, CA) using a Phoenix crystallization robot (Art Robbins Instruments) and commercially available crystallization screens. The drops were composed of equal volumes of protein and reservoir buffer.
Diffraction quality crystals were harvested directly from the original sitting drop plates from the following crystallization solutions: A141E: 0.1 M BIS-TRIS pH 6.5; 25% w/v PEG 3350 at a protein concentration of 18.4 mg/ml. A141D: 200 mM sodium chloride; 0.1 M BIS-TRIS pH 5.5; 25% w/v PEG 3350 at a protein concentration of 9 mg/ml. All crystals harvested for X-ray analysis were flash-cooled in liquid nitrogen, and diffraction experiments were performed on a beamline B14-1 at Stanford Synchrotron Radiation Lightsource (Menlo Park, CA) at 100K. Diffraction data collected from a single crystal for each Fab were processed, integrated, and scaled with XDS software (Kabsch, 2010).
Structures of both Fab molecules were determined using molecular replacement method with program MolRep (Vagin, 1997) from CCP4 (Winn, 2011) suite of crystallographic software. Model building was performed using Coot (Emsley, 2004), for refinement program Refmac5 (Kovalevskiy, 2018) was used.
Crystal of T117R/A141D Fab diffracted to 2.1 Å. Upon completion of the refinement we found that in line with our prediction side chains of mutated amino acids indeed established quite strong hydrogen bond (
Crystal of T117R/A141E Fab diffracted to 2.0 Å. Upon completion of the refinement we found that in line with our prediction side chains of mutated amino acids indeed established hydrogen bond.
Comparison of these two Fab molecules show that mutations T117R/A141D establish stronger (shorter) hydrogen bond than T117R/A141E. This result has been confirmed by higher percentage of correct paired molecules for 117R/141D pair containing molecules.
Following substitutions are underlined:
Engineered disulfide: S122C, C212V
Following substitutions are underlined:
Engineered disulfide: F126C, C220V
Following substitutions are underlined:
“Hole” mutations (T366S, L368A, and Y407V).
Following substitutions are underlined:
Hole” mutations (T366S, L368A, and Y407V); and stabilizing cysteine mutation (Y349C).
Following substitutions are underlined:
“Knob” mutation (T366W).
Following substitutions are underlined:
“Knob” mutation (T366W); stabilizing cysteine mutation (S354C).
Following substitutions are underlined:
Hole” mutations (T366S, L368A, and Y407V); and stabilizing cysteine mutation (Y349C).
Following substitutions are underlined:
“Knob” mutation (T366W); stabilizing cysteine mutation (S354C).
Following substitutions are underlined:
“Knob” mutation (T366W); interchain cysteine mutations (F126C and C220V); stabilizing cysteine mutation (S354C).
This application claims the benefit of U.S. Provisional Patent Application No. 63/494,610, filed Apr. 6, 2023, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63494610 | Apr 2023 | US |
