Multispecific antigen-binding proteins and uses thereof
Multispecific antigen-binding proteins with CD3 and CAIX targeting domains, combined with half-life extension units, address the limitations of existing antibodies by improving stability and efficacy in treating solid tumors.
Patent Information
- Application Number
- PCT/CN2025/082732
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-03-16
- Filing Date
- 2025-03-14
- Publication Date
- 2025-09-25
AI Technical Summary
Existing multispecific antibodies face challenges such as high on-target off-tumor toxicity, severe cytokine release syndrome, and difficulties in expression and purification, limiting their efficacy in treating solid tumors.
Development of multispecific antigen-binding proteins that include a T cell binding domain for CD3 and a tumor-associated antigen binding domain for CAIX, with a half-life extension unit and cleavable linkers to enhance stability and specificity, allowing simultaneous targeting of both antigens.
The multispecific antigen-binding proteins effectively redirect T cells to tumor cells, reducing off-tumor toxicity and enhancing therapeutic efficacy while maintaining stability and specificity.
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Abstract
Description
MULTISPECIFIC ANTIGEN-BINDING PROTEINS AND USES THEREOF
[0001] CLAIM OF PRIORITY
[0002] This application claims the benefit of International Patent Application App. No. PCT / CN2024 / 082070, filed on March 16, 2024, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD
[0003] This disclosure relates to multispecific antigen-binding proteins and uses thereof.BACKGROUND
[0004] Naturally occurring antibodies typically only target one antigen. A multispecific antibody can be manufactured in different structural formats, so that they can simultaneously bind to two or more different epitopes. These epitopes can be in the same antigen or in different antigens. This opens up a wide range of applications, including redirecting T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators, and delivering payloads to targeted sites.
[0005] The approval of Catumaxomab, the first T cell engager (TCE) for solid tumor in 2009 brought hope that TCEs could achieve both high responses and long benefits in solid tumor patients. However, over the following 15 years, efforts to develop TCEs for solid tumors resulted in the approval of only two additional drugs: the TCR-based Tebentafusp and the antibody-based Tarlatamab. The limited progress in this field is largely attributed to challenges such as high on-target off-tumor toxicity and severe cytokine release syndrome (CRS) , which contribute not only to adverse effects but also to reduced efficacy. In some cases, a multispecific antibody may not have the desired efficacy and it can be difficult to express and purify. Thus, there is a need to continue to develop various therapeutics based on multispecific antibodies or antigen-binding proteins.SUMMARY
[0006] This disclosure relates to multispecific antigen-binding proteins, wherein the multispecific antigen-binding proteins specifically bind to a T cell antigen (e.g., CD3) and / or one or more tumor-associated antigens (e.g., CAIX) , or a combination thereof. In one aspect, the disclosure is related to a multispecific antigen-binding proteins that comprise a T cell binding domain that specifically binds to a T cell antigen (e.g. CD3) and a CAIX-binding domain that specifically binds to CAIX.
[0007] In one aspect, the disclosure is related to an antigen-binding protein, comprising: (a) a first antigen-binding domain (B1) comprising a Fab and a first mask peptide (M1) , in some embodiments, the Fab comprises a first chain and a second chain, in some embodiments, the first mask peptide (M1) impairs the binding of the first antigen-binding domain (B1) to the target of the first antigen-binding domain (B1) , and the first mask peptide (M1) is linked to the first antigen-binding domain (B1) via a first cleavable linker (L1) ; (b) a second antigen-binding domain (B2) , in some embodiments, the second antigen-binding domain (B2) comprises a first VHH that is linked to C-terminus of the first or second chain of the Fab; and (c) a half-life extension unit (E) , in some embodiments, the half-life extension unit (E) is linked to N-terminus of the first mask peptide (M1) . In some embodiments, (1) the first chain of the Fab comprises a heavy chain variable region (VH) and a CH1 domain and the second chain of the Fab comprises a light chain variable region (VL) and a CL domain; (2) the first chain of the Fab comprises a VL and a CL domain and the second chain of the Fab comprises a VH and a CH1 domain; (3) the first chain of the Fab comprises a VH and a CL domain and the second chain of the Fab comprises a VL and a CH1 domain; or (4) the first chain of the Fab comprises a VL and a CH1 domain and the second chain of the Fab comprises a VH and a CL domain. In some embodiments, the first cleavable linker (L1) is linked to N-terminus of the VH or the VL. In some embodiments, the antigen-binding further comprises a second VHH, optionally the first and second VHHs are the same. In some embodiments, (1) the first VHH is linked to C-terminus of the first chain of the Fab and the second VHH is linked to C-terminus of second chain of the Fab; (2) the first VHH is linked to C-terminus of the second chain of the Fab and the second VHH is linked to C-terminus of first chain of the Fab; (3) the first VHH is linked to the C-terminus of the first chain of the Fab and the second VHH is linked to the C-terminus of the first VHH; or (4) the first VHH is linked to the C-terminus of the second chain of the Fab and the second VHH is linked to the C-terminus of the first VHH.
[0008] In some embodiments, the first antigen-binding domain (B1) specifically binds to an immune cell receptor antigen (e.g., CD3, 4-1BB (CD137) , 4-1BB-L, CD28, CD40, CD40-L, CD58, CD2, or CD8) and the second antigen-binding domain specifically binds to a tumor antigen (e.g., CAIX) .
[0009] In one aspect, the disclosure is related to an antigen-binding protein, comprising: (a) a first antigen-binding domain (B1) comprising a Fab and a first mask peptide (M1) , wherein the Fab comprises a first chain and a second chain, wherein the first mask peptide (M1) impairs the binding of the first antigen-binding domain (B1) to the target of the first antigen-binding domain (B1) , and the first mask peptide (M1) is linked to the first antigen-binding domain (B1) via a first cleavable linker (L1) ; (b) a second antigen-binding domain (B2) , wherein the second antigen-binding domain (B2) comprises a first VHH that is linked to C-terminus of the first or second chain of the Fab; and (c) a half-life extension unit (E) , wherein the half-life extension unit (E) is linked to N-terminus of the first mask peptide (M1) .
[0010] In some embodiments, (1) the first chain of the Fab comprises a heavy chain variable region (VH) and a CH1 domain and the second chain of the Fab comprises a light chain variable region (VL) and a CL domain; (2) the first chain of the Fab comprises a VL and a CL domain and the second chain of the Fab comprises a VH and a CH1 domain; (3) the first chain of the Fab comprises a VH and a CL domain and the second chain of the Fab comprises a VL and a CH1 domain; or (4) the first chain of the Fab comprises a VL and a CH1 domain and the second chain of the Fab comprises a VH and a CL domain.
[0011] In some embodiments, the first cleavable linker (L1) is linked to N-terminus of the VH or the VL.
[0012] In some embodiments, the antigen-binding further comprises a second VHH, optionally the first and second VHHs are the same.
[0013] In some embodiments, (1) the first VHH is linked to C-terminus of the first chain of the Fab and the second VHH is linked to C-terminus of second chain of the Fab; (2) the first VHH is linked to C-terminus of the second chain of the Fab and the second VHH is linked to C-terminus of first chain of the Fab; (3) the first VHH is linked to the C-terminus of the first chain of the Fab and the second VHH is linked to the C-terminus of the first VHH; or (4) the first VHH is linked to the C-terminus of the second chain of the Fab and the second VHH is linked to the C-terminus of the first VHH.
[0014] In some embodiments, the first antigen-binding domain (B1) specifically binds to an immune cell receptor antigen (e.g., CD3, 4-1BB (CD137) , 4-1BB-L, CD28, CD40, CD40-L, CD58, CD2, or CD8) and the second antigen-binding domain (B2) specifically binds to a tumor antigen (e.g., CAIX) .
[0015] In some embodiments, first antigen-binding domain (B1) specifically binds to CD3, and comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises any one of the combinations of the HCDR1, HCDR2, and HCDR3 sequences in Tables 1, 3, and 5, and the VL comprises any one of the combinations of the LCDR1, LCDR2, and LCDR3 sequences in Tables 2, 4, and 6.
[0016] In some embodiments, first antigen-binding domain (B1) specifically binds to CD3, and comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises any one of the amino acid sequences in Table 7, and the VL comprise any one of the amino sequences in Table 8.
[0017] In some embodiments, the second antigen-binding domain (B2) specifically binds to CAIX, wherein the second antigen-binding domain (B2) comprises a VHH comprising any one of the combinations of the CDR1, CDR2, and CDR3 sequences in Tables 15-17.
[0018] In some embodiments, the first antigen-binding domain (B1) specifically binds to CD3, and comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein:
[0019] (1) the VH comprises, according to the Kabat definition:
[0020] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 1, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0021] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 2, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0022] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0023] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0024] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 4, 6, and 8, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0025] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 9, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0026] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 10, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0027] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 11, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0028] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 12, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0029] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 8, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0030] the VL comprises, according to the Kabat definition:
[0031] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 13, 19, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0032] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 14, 20, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0033] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 13, 20, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0034] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0035] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 16, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0036] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 17, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0037] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 18, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0038] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 22, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0039] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 23, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0040] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 24, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0041] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 25, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0042] (2) the VH comprises, according to the Chothia definition:
[0043] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 29, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0044] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 30, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0045] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 31, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0046] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 32, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0047] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 33, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0048] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 34, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0049] the VL comprises, according to the Chothia definition:
[0050] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 35, 41, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0051] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 36, 42, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0052] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 35, 42, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0053] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0054] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 38, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0055] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 39, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0056] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 40, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0057] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 44, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0058] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 45, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0059] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 46, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0060] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 47, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0061] (3) the VH comprises, according to the IMGT definition:
[0062] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 52, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0063] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 50, 51, and 53, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0064] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 54, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0065] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 55, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0066] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 56, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0067] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 57, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0068] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 53, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0069] the VL comprises, according to the IMGT definition:
[0070] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 58, 60, and 61, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0071] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 59, 60, and 61, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) .
[0072] In some embodiments, the first antigen-binding domain (B1) comprises a VH and a VL, and:
[0073] (1) according to the kabat definition:
[0074] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 26;
[0075] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 2, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 26;
[0076] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 14, LC-CDR2: SEQ ID NO: 20, and LC-CDR3: SEQ ID NO: 26;
[0077] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 20, and LC-CDR3: SEQ ID NO: 26;
[0078] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0079] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 16, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0080] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 8; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0081] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 9; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0082] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 10; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0083] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 11; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0084] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0085] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 17, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0086] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 18, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0087] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 26;
[0088] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 23, and LC-CDR3: SEQ ID NO: 26;
[0089] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 24, and LC-CDR3: SEQ ID NO: 26;
[0090] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 25, and LC-CDR3: SEQ ID NO: 26; or
[0091] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 8; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26; or
[0092] (2) according to the chothia definition:
[0093] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 35, LC-CDR2: SEQ ID NO: 41, and LC-CDR3: SEQ ID NO: 48;
[0094] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 36, LC-CDR2: SEQ ID NO: 42, and LC-CDR3: SEQ ID NO: 48;
[0095] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 35, LC-CDR2: SEQ ID NO: 42, and LC-CDR3: SEQ ID NO: 48;
[0096] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0097] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 38, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0098] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 30; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0099] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 31; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0100] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 32; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0101] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 33; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0102] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0103] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 39, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0104] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 40, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0105] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 44, and LC-CDR3: SEQ ID NO: 48;
[0106] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 45, and LC-CDR3: SEQ ID NO: 48;
[0107] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 46, and LC-CDR3: SEQ ID NO: 48; or
[0108] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 47, and LC-CDR3: SEQ ID NO: 48; or
[0109] (3) according to the IMGT definition:
[0110] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 52; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0111] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 52; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 59, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0112] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 50, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 53; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0113] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 54; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0114] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 55; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0115] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 56; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0116] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 57; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61; or
[0117] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 53; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61.
[0118] In some embodiments, the second antigen-binding domain (B2) specifically binds to CAIX, wherein the second antigen-binding domain (B2) comprises a VHH comprising:
[0119] (1) according to the kabat definition:
[0120] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 167, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0121] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 168, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0122] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 169, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0123] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 170, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0124] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 171, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0125] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 172, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0126] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 173, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0127] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 174, 180, and 186, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0128] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 175, 181, and 187, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0129] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 171, 182, and 188, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0130] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 176, 183, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0131] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 183, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0132] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 178, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0133] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0134] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 190, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0135] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 191, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0136] (2) according to the chothia definition:
[0137] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 192, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0138] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 193, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0139] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 194, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0140] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 195, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0141] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 196, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0142] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 197, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0143] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 198, 203, and 208, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0144] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 199, 204, and 209, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0145] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 195, 205, and 210, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0146] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 200, 206, and 211, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0147] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 211, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0148] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 212, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0149] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 213, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0150] (3) according to the IMGT definition:
[0151] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 214, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0152] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 215, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0153] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 216, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0154] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 217, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0155] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 218, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0156] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 219, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0157] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 220, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0158] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 221, 227, and 232, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0159] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 222, 228, and 233, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0160] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 218, 229, and 234, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0161] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 223, 230, and 235, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0162] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 224, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0163] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 235, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0164] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 236, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0165] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 237, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) .
[0166] In some embodiments, the second antigen-binding domain (B2) comprises a VHH that comprises: (a) the amino acid sequence of any one of SEQ ID NOs: 238-257; (b) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 238-257; or (c) an amino acid sequence that has one or more additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 238-257, wherein the additions, deletions and / or substitutions do not occur in a CDR region.
[0167] In some embodiments, the first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.
[0168] In some embodiments, the first mask peptide (M1) is linked to the half-life extension unit (E) via a linker that comprises the amino acid sequence of any one of SEQ ID NOs: 273-288.
[0169] In some embodiments, the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.
[0170] In some embodiments, the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .
[0171] In some embodiments, the VHH that specifically binds to human serum albumin (HSA) comprises the amino acid sequence of SEQ ID NO: 268.
[0172] In one aspect, the disclosure is related to an antigen-binding protein comprising: (1) a CD3-binding domain, and (2) a CAIX-binding domain, wherein the CD3-binding domain comprises a Fab comprising a heavy chain variable region (VH) and a light chain variable region (VL) , and the CAIX-binding domain comprises a VHH, wherein the CAIX-binding domain is linked to C-terminus of the CD3-binding domain.
[0173] In some embodiments, first antigen-binding domain (B1) specifically binds to CD3, and comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises any one of the combinations of the HCDR1, HCDR2, and HCDR3 sequences in Tables 1, 3, and 5, and the VL comprises any one of the combinations of the LCDR1, LCDR2, and LCDR3 sequences in Tables 2, 4, and 6.
[0174] In some embodiments, first antigen-binding domain (B1) specifically binds to CD3, and comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH comprises any one of the amino acid sequences in Table 7, and the VL comprise any one of the amino sequences in Table 8.
[0175] In some embodiments, the CAIX-binding domain comprises a VHH comprising any one of the combinations of the CDR1, CDR2, and CDR3 sequences in Tables 15-17.
[0176] In some embodiments, for the first binding domain (B1) , the VH comprises, according to the Kabat definition:
[0177] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 1, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0178] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 2, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0179] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 5, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0180] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 7, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0181] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 4, 6, and 8, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0182] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 9, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0183] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 10, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0184] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 11, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0185] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 12, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0186] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 3, 6, and 8, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0187] the VL comprises, according to the Kabat definition:
[0188] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 13, 19, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0189] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 14, 20, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0190] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 13, 20, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0191] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0192] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 16, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0193] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 17, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0194] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 18, 21, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0195] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 22, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0196] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 23, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0197] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 24, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0198] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 15, 25, and 26, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0199] In some embodiments, the VH comprises, according to the Chothia definition:
[0200] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 29, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0201] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 30, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0202] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 31, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0203] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 32, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0204] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 33, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0205] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 27, 28, and 34, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0206] the VL comprises, according to the Chothia definition:
[0207] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 35, 41, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0208] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 36, 42, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0209] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 35, 42, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0210] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0211] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 38, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0212] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 39, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0213] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 40, 43, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0214] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 44, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0215] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 45, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0216] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 46, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0217] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 37, 47, and 48, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0218] In some embodiments, the VH comprises, according to the IMGT definition:
[0219] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 52, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0220] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 50, 51, and 53, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0221] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 54, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0222] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 55, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0223] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 56, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0224] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 57, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0225] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 49, 51, and 53, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; and
[0226] the VL comprises, according to the IMGT definition:
[0227] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 58, 60, and 61, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0228] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 59, 60, and 61, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) .
[0229] In some embodiments, according to the kabat definition:
[0230] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 26;
[0231] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 2, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 26;
[0232] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 14, LC-CDR2: SEQ ID NO: 20, and LC-CDR3: SEQ ID NO: 26;
[0233] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 13, LC-CDR2: SEQ ID NO: 20, and LC-CDR3: SEQ ID NO: 26;
[0234] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0235] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 7; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 16, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0236] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 8; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0237] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 9; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0238] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 10; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0239] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 11; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0240] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0241] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 17, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0242] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 18, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26;
[0243] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 26;
[0244] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 23, and LC-CDR3: SEQ ID NO: 26;
[0245] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 24, and LC-CDR3: SEQ ID NO: 26;
[0246] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 12; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 25, and LC-CDR3: SEQ ID NO: 26; or
[0247] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 3, HC-CDR2: SEQ ID NO: 6, and HC-CDR3: SEQ ID NO: 8; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 21, and LC-CDR3: SEQ ID NO: 26; or
[0248] In some embodiments, according to the chothia definition:
[0249] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 35, LC-CDR2: SEQ ID NO: 41, and LC-CDR3: SEQ ID NO: 48;
[0250] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 36, LC-CDR2: SEQ ID NO: 42, and LC-CDR3: SEQ ID NO: 48;
[0251] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 35, LC-CDR2: SEQ ID NO: 42, and LC-CDR3: SEQ ID NO: 48;
[0252] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0253] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 29; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 38, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0254] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 30; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0255] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 31; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0256] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 32; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0257] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 33; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0258] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0259] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 39, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0260] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 40, LC-CDR2: SEQ ID NO: 43, and LC-CDR3: SEQ ID NO: 48;
[0261] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 44, and LC-CDR3: SEQ ID NO: 48;
[0262] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 45, and LC-CDR3: SEQ ID NO: 48;
[0263] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 46, and LC-CDR3: SEQ ID NO: 48; or
[0264] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 27, HC-CDR2: SEQ ID NO: 28, and HC-CDR3: SEQ ID NO: 34; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 37, LC-CDR2: SEQ ID NO: 47, and LC-CDR3: SEQ ID NO: 48; or
[0265] In some embodiments, according to the IMGT definition:
[0266] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 52; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0267] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 52; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 59, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0268] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 50, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 53; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0269] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 54; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0270] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 55; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0271] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 56; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61;
[0272] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 57; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61; or
[0273] the first antigen-binding domain (B1) comprises complementarity determining regions (CDRs) : HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the first antigen-binding domain (B1) comprise: HC-CDR1: SEQ ID NO: 49, HC-CDR2: SEQ ID NO: 51, and HC-CDR3: SEQ ID NO: 53; and the first antigen-binding domain (B1) comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the first antigen-binding domain (B1) comprise LC-CDR1: SEQ ID NO: 58, LC-CDR2: SEQ ID NO: 60, and LC-CDR3: SEQ ID NO: 61.
[0274] In some embodiments, the CAIX-binding domain comprises a VHH comprising:
[0275] (1) according to the kabat definition:
[0276] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 167, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0277] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 168, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0278] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 169, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0279] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 170, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0280] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 171, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0281] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 172, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0282] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 173, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0283] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 174, 180, and 186, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0284] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 175, 181, and 187, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0285] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 171, 182, and 188, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0286] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 176, 183, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0287] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 183, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0288] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 178, 179, and 185, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0289] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 189, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0290] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 190, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0291] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 177, 184, and 191, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0292] (2) according to the chothia definition:
[0293] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 192, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0294] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 193, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0295] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 194, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0296] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 195, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0297] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 196, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0298] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 197, 202, and 207, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0299] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 198, 203, and 208, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0300] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 199, 204, and 209, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0301] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 195, 205, and 210, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0302] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 200, 206, and 211, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0303] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 211, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0304] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 212, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0305] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 201, 206, and 213, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0306] (3) according to the IMGT definition:
[0307] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 214, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0308] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 215, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0309] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 216, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0310] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 217, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0311] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 218, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0312] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 219, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0313] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 220, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0314] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 221, 227, and 232, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0315] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 222, 228, and 233, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0316] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 218, 229, and 234, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0317] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 223, 230, and 235, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0318] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 224, 226, and 231, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0319] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 235, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ;
[0320] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 236, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) ; or
[0321] CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NOs: 225, 230, and 237, or variant sequences thereof having one or more amino acids substituted, deleted, or added (e.g., one, two, or three amino acids substituted, deleted, or added) .
[0322] In some embodiments, the CAIX-binding domain comprises a VHH that comprises: (a) the amino acid sequence of any one of SEQ ID NOs: 238-257; (b) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 238-257; or (c) an amino acid sequence that has one or more additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 238-257, wherein the additions, deletions and / or substitutions do not occur in a CDR region.
[0323] In some embodiments, the antigen-binding protein further comprises a first mask peptide (M1) that impairs the binding of the CD3-binding domain to the target of the CD3-binding domain (e.g., CD3) , and the first mask peptide is linked to the CD3-binding domain via a first cleavable linker (L1) .
[0324] In some embodiments, first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.
[0325] In some embodiments, the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.
[0326] In some embodiments, the antigen-binding protein further comprises a half-life extending unit (E) .
[0327] In some embodiments, the half-life extending unit (E) is linked to the N-terminus of the first mask peptide (M1) via a linker that comprises the amino acid sequence of any one of SEQ ID NOs: 273-288.
[0328] In some embodiments, the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .
[0329] In some embodiments, the VHH that specifically binds to human serum albumin (HSA) comprises the amino acid sequence of SEQ ID NO: 268.
[0330] In one aspect, the disclosure is related to an antigen-binding protein, comprising: a first peptide comprising, from the N-terminus to the C-terminus, a half-life extension unit (E) , a first mask peptide (M1) , a first cleavable linker (L1) , a VH, a CH1, a first VHH that specifically binds to a first antigen; and a second peptide comprising, from the N-terminus to the C-terminus, a VL, and a CL, wherein the VH and VL form a Fab that specifically binds to a second antigen, and the first mask peptide (M1) impairs the binding of the Fab to the second antigen.
[0331] In some embodiments, the first peptide further comprises a second VHH that is linked to the C-terminus of the first VHH.
[0332] In some embodiments, the first and second VHHs are the same.
[0333] In some embodiments, the first antigen is CAIX and the second antigen is CD3.
[0334] In some embodiments, the VH comprises any one of the combinations of the HCDR1, HCDR2, and HCDR3 sequences in Tables 1, 3, and 5, and the VL comprises any one of the combinations of the LCDR1, LCDR2, and LCDR3 sequences in Tables 2, 4, and 6.
[0335] In some embodiments, the VH comprises any one of the amino acid sequences in Table 7, and the VL comprise any one of the amino sequences in Table 8.
[0336] In some embodiments, the first VHH comprises any one of the combinations of the CDR1, CDR2, and CDR3 sequences in Tables 15-17.
[0337] In some embodiments, the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .
[0338] In some embodiments, the first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.
[0339] In some embodiments, the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.
[0340] In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the antigen-binding protein described herein.
[0341] In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA) .
[0342] In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein.
[0343] In one aspect, the disclosure is related to a cell comprising the vector described herein.
[0344] In some embodiments, the cell is a HEK293 cell or CHO cell.
[0345] In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids described herein.
[0346] In one aspect, the disclosure is related to a method of producing an antigen-binding protein, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the antigen-binding protein; and (b) collecting the antigen-binding protein produced by the cell.
[0347] In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antigen-binding protein described herein, to the subject.
[0348] In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein described herein.
[0349] In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein described herein.
[0350] In one aspect, the disclosure is related to a pharmaceutical composition comprising the antigen-binding protein described herein, and a pharmaceutically acceptable carrier.
[0351] In one aspect, the disclosure is related to a chimeric antigen receptor (CAR) comprising the antigen-binding protein described herein.
[0352] In one aspect, the disclosure is related to an antibody-drug conjugate (ADC) comprising the antigen-binding protein described herein, covalently bound to a therapeutic agent.
[0353] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.
[0354] In one aspect, the disclosure is related to a pharmaceutical composition comprising the antigen-binding protein described herein, the CAR described herein, or the antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
[0355] In one aspect, the disclosure is related to a method of treating a subject having a disease, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition described herein to the subject.
[0356] In some embodiments, the disease is cancer; preferably, the cancer is selected from the group consisting of KIRC (Kidney Renal Clear Cell Carcinoma) , CHOL (Cholangio carcinoma) , PAAD (Pancreatic adenocarcinoma) , LUSC (Lung squamous cell carcinoma) , LUAD (Lung adenocarcinoma) , HNSC (Head and Neck squamous cell carcinoma) , BLCA (Bladder Urothelial Carcinoma) , ESCA (Esophageal carcinoma) , COAD (Colon adenocarcinoma) , READ (Rectum adenocarcinoma) , SCLC (Small cell lung cancer) , STAD (Stomach adenocarcinoma) , MESO (Mesothelioma) and CESC (Cervical squamous cell carcinoma) .
[0357] In some embodiments, the subject has a CAIX-positive cancer, e.g., cervical cancer, renal cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, or bladder cancer.
[0358] In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the antigen-binding protein described herein. In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA) .
[0359] In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein.
[0360] In one aspect, the disclosure is related to a cell comprising the vector described herein. In some embodiments, the cell is a HEK293 cell or CHO cell. In one aspect, the disclosure is related to a cell comprising one or more of the nucleic acids described herein.
[0361] In one aspect, the disclosure is related to a method of producing an antigen-binding protein, the method comprising (a) culturing the cell described herein under conditions sufficient for the cell to produce the antigen-binding protein; and (b) collecting the antigen-binding protein produced by the cell.
[0362] In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antigen-binding protein described herein, to the subject.
[0363] In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein described herein.
[0364] In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein described herein.
[0365] In one aspect, the disclosure is related to a pharmaceutical composition comprising the antigen-binding protein described herein, and a pharmaceutically acceptable carrier.
[0366] In one aspect, the disclosure is related to a chimeric antigen receptor (CAR) comprising the antigen-binding protein described herein.
[0367] In one aspect, the disclosure is related to an antibody-drug conjugate (ADC) comprising the antigen-binding protein described herein, covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.
[0368] In one aspect, the disclosure is related to a pharmaceutical composition comprising the antigen-binding protein, the CAR, or the antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
[0369] In one aspect, the disclosure is related to a method of treating a subject having a disease, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition described herein to the subject. In some embodiments, the disease is cancer; preferably, the cancer is selected from the group consisting of KIRC (Kidney Renal Clear Cell Carcinoma) , CHOL (Cholangio carcinoma) , PAAD (Pancreatic adenocarcinoma) , LUSC (Lung squamous cell carcinoma) , LUAD (Lung adenocarcinoma) , HNSC (Head and Neck squamous cell carcinoma) , BLCA (Bladder Urothelial Carcinoma) , ESCA (Esophageal carcinoma) , COAD (Colon adenocarcinoma) , READ (Rectum adenocarcinoma) , SCLC (Small cell lung cancer) , STAD (Stomach adenocarcinoma) , MESO (Mesothelioma) and CESC (Cervical squamous cell carcinoma) . In some embodiments, the subject has a CAIX-positive cancer, e.g., cervical cancer, renal cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, or bladder cancer.
[0370] As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope in an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, single variable domain (VHH) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., multispecific antibodies, bispecific antibodies, trispecific antibodies, single-chain antibodies, diabodies, linear antibodies formed from these antibodies or antibody fragments, and antigen-binding protein constructs.
[0371] As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a VHH) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’) 2, and Fv fragments, scFv, and VHH.
[0372] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
[0373] As used herein, when referring to an antibody or an antigen-binding fragment, the phrases “specifically binding” and “specifically binds” mean that the antibody or an antigen-binding fragment interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to CAIX may be referred to as CAIX-specific antibody or an anti-CAIX antibody.
[0374] As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.
[0375] As used herein, the term “trispecific antibody” refers to an antibody that binds to three different epitopes. The epitopes can be on the same antigen or on different antigens.
[0376] As used herein, the term “multispecific antibody” refers to an antibody that binds to two or more different epitopes. The epitopes can be on the same antigen or on different antigens. A multispecific antibody can be e.g., a bispecific antibody or a trispecific antibody. In some embodiments, the multispecific antibody binds to two, three, four, five, or six different epitopes.
[0377] As used herein, a “VHH” refers to the variable domain of a heavy chain antibody. In some embodiments, the VHH is a humanized VHH. In some embodiments, the VHH is a single-domain antibody (sdAb) .
[0378] As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
[0379] As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA / RNA hybrids, and modifications thereof.
[0380] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
[0381] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.DESCRIPTION OF DRAWINGS
[0382] FIG. 1 shows schematic structure of Structures 1-1 to 1-13.
[0383] FIG. 2 shows schematic structures of the CD3 / CAIX bispecific antibody ( “CD3 / CAIX-BsAb” ) including an anti-CD3 Fab and an anti-CAIX VHH.
[0384] FIGS. 3-4 show schematic structures of several CD3 / CAIX / CAIX multispecific antibody constructs including (1) an anti-CD3 Fab, (2) a first anti-CAIX VHH and (3) a second anti-CAIX VHH.
[0385] FIG. 5 shows schematic structure of Structures 5-1A to 5-9I.DETAILED DESCRIPTION
[0386] This disclosure relates to multispecific antibodies or antigen-binding fragments thereof, and antigen-binding proteins (e.g., bispecific or trispecific antigen-binding proteins) . In one aspect, the multispecific antibodies or antigen-binding fragments thereof, or antigen-binding proteins can bind to a T cell antigen (e.g., CD3) and / or one or more tumor-associated antigens (e.g., CAIX) , or a combination thereof.
[0387] Cluster of differentiation 3 (CD3) is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p 172 and 178, 1999) . In mammals, the complex comprises a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimer of CD3ζchains. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζchain has three. It is believed the ITAMs are important for the signaling capacity of a TCR complex.
[0388] Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme that is overexpressed by cancer cells from many tumor types, and is a component of the pH regulatory system invoked by these cells to combat the deleterious effects of a high rate of glycolytic metabolism. CAIX is constitutively overexpressed in the vast majority of clear cell renal cell carcinoma (ccRCC) and can also be induced in hypoxic microenvironments, a major hallmark of most solid tumors. CAIX expression is restricted to a few sites in healthy tissues, positioning this molecule as a strategic target for cancer immunotherapy. Carbonic anhydrases are metalloenzymes that reversibly catalyze the hydration of carbon dioxide, generating bicarbonate ions and protons. Several tumors, such as clear cell renal cell carcinoma (ccRCC) , glioblastoma, triple-negative breast cancer, ovarian cancer, colorectal, and others overexpress carbonic anhydrase isoform IX (CAIX) . This transmembrane enzyme differs from most other CAs by having its catalytic site located in the extracellular domain, responsible for tumor microenvironment acidification. In consequence of the low pH, cathepsin B and other proteolytic enzymes are activated, creating a favorable environment for cancer cell migration and metastasis. An acidic pH also impairs the tumoricidal function of cytotoxic T cells and natural killer cells (NK) , favoring the occurrence of minimal residual disease and recurrence.
[0389] Solid tumors rely primarily on the blood supply to deliver oxygen and nutrients to help them grow. However, as tumors grow, blood vessels are unable to provide oxygen and nutrients to every part of the tumor, resulting in localized hypoxia. Hypoxia initiates a global adaptive response, mainly by activating hypoxia-inducible factor (HIF) to trigger metabolic shifts that are critical for cell survival to increase reliance on the glycolytic pathway. This will lead to the continuous accumulation of acidic by-products and reliance on pH-regulating enzymes and transporters to maintain intracellular alkaline pH to maintain tumor cell proliferation and survival. The extracellular environment becomes acidic due to increased lactate and proton efflux. Acidic pH also causes metabolic reorganization to meet the cell's macromolecule and bioenergy needs and maintain its own survival.
[0390] Carbonic anhydrase (CA) is a type of metalloprotease that catalyzes the reversible hydration of CO2 into bicarbonate and protons
[0391] CAIX is a tumor-associated cell surface glycoprotein that is induced by hypoxia and is anchored in the plasma membrane via a single transmembrane domain and a short cytoplasmic tail. Its active center faces the outside of the cell and contributes to CO2 hydration and pH regulation, while promoting CO2 diffusion and proton migration in tumor cells, helping the adaptation to acidosis.
[0392] The main function of CAIX is to maintain intracellular pH homeostasis under hypoxic conditions. Overexpression of CAIX in tumor cells is considered a hypoxic marker of the tumor microenvironment and is closely related to poor prognosis of patients. It is now generally believed that CAIX is involved in tumor formation and progression.
[0393] On the one hand, CAIX can cooperate with bicarbonate transporters (NBC) and monocarboxylate transporters (MCT) to remove acid from cells and ensure that alkaline pH is maintained in cells, thus benefiting survival of tumor cells. CAIX catalyzes the conversion of extracellular CO2 into protons and bicarbonate ions through its extracellular active center. Bicarbonate ions are transported from the adjacent NBC to the cytoplasmic matrix. In the cell, bicarbonate is catalyzed by CA II in the cytoplasm matrix and combines with protons to undergo a reverse reaction and is converted into CO2, which leaves the cell through diffusion. On the other hand, the protons generated by the CAIX catalyzed reaction remain outside the cell and contribute to the acidification of the surrounding environment of the cell, supporting tumor cells to invade surrounding tissues and eventually become a more aggressive form of tumor capable of spreading to other organs.
[0394] The CAIX gene is located downstream of the von Hippel-Lindau (VHL) tumor suppressor gene and is activated by the HIF1 pathway. VHL is a ubiquitin ligase (E3) . Under normoxic conditions, HIF1 / 2α is hydroxylated. VHL targets the hydroxylated HIF1 / 2α to cause ubiquitination and degradation. VHL protein controls the expression of CAIX by degrading HIF protein. Inactivation of VHL is very common in clear cell renal cell carcinoma (ccRCC) . It has been reported that inactivating mutations of VHL exist in up to 90%of ccRCC. In ccRCC, inactivating mutations in VHL lead to the loss of function of the VHL tumor suppressor gene, leading to the accumulation of HIF1 / 2α, thereby upregulating the expression of CAIX.
[0395] Common tumor types overexpressing CAIX include cervical cancer, renal cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, bladder cancer, etc. In normal tissues, CAIX is mainly expressed in epithelial cells of the stomach, bile duct, and small intestine. However, unlike tumor cells, CAIX expressed in normal tissues is mainly located in the cytoplasm.
[0396] Immunohistochemistry studies have shown that more than 85%of primary renal cancers and metastatic renal cancers express CAIX antigen, and almost all ccRCC, which accounts for 90%of renal cancers, express CAIX antigen, while normal renal tissue does not express CAIX antigen. Therefore, CAIX is an ideal target for tumor therapy.
[0397] In general, multispecific antibodies (e.g., trispecific antibodies) include two or more antigen-binding sites targeting different antigens or different epitopes of the same antigen. Thus, multispecific antibodies (e.g., trispecific antibodies) can have more functions than a monospecific antibody. For example, these functions include, but not limited to, stronger binding to an antigen through an avidity effect; co-localization of bound antigens on the cell surface and the effect therefrom; increasing the serum half-life of an antibody fragment by linking it to a antibody fragment that is bound to a protein with a long serum half-life, e.g., albumin or transferrin; and bringing two cells into proximity by binding to an antigen on each of the cells.
[0398] Among the purposes of multispecific antibodies (e.g., trispecific antibodies) , one class of molecules, T cell engagers (TCE) , has gained more attention. A TCE is usually a multispecific antibody (e.g., trispecific antibodies) which binds to an antigen on a T cell and an antigen on another cell simultaneously. CD3 is usually selected as the antigen on the T cell. A cancer or tumor cell is usually selected as the other cell type as discussed above. Through binding to CD3 on T cells and a tumor associated antigen (TAA) on cancer cells, the TCE can induce activation of T cells upon binding to cancer cells and cause the killing of the latter.
[0399] Nevertheless, there are some hurdles to overcome in order to generate desirable homogeneous multispecific antibodies (e.g., trispecific antibodies) . The first hurdle is mismatch of heavy chains that bind to the same target (e.g., antigen or epitope) . For example, to generate multispecific antibodies with a desired format, the heavy chains targeting different targets should ideally form a heterodimer. However, the percentage of the desired multispecific antibodies varies greatly in different constructs.
[0400] The first hurdle to overcome is the mismatch between heavy chain variable regions (VHs) and light chain variable regions (VLs) . A monoclonal antibody has two identical Fab fragments, each having a paired VH and VL. By contrast, a multispecific antibody usually has several different heavy chain variable regions and several different light chain variable regions. Therefore, there is a possibility that each VH can bind to multiple VLs and each VL can bind to multiple VHs. As a result, only some of the formed multispecific antibodies are functional without addressing this mismatch issue.
[0401] Several strategies have been designed to disable this mismatch. One solution is to design an antibody with a common light chain. Specifically, a light chain, or more precisely a VL, is selected which can form a dimer with all VHs. This design abrogated the necessity of matching VH and VL with the same target-binding specificity. The shortcoming of this strategy is that the contribution of VL in target binding is greatly reduced, leading to difficulty finding an optimal VH as the VH will be greatly if not entirely responsible for target binding.
[0402] Another solution is the use of CrossMAb technology, in which a VL is fused to heavy chain constant domain 1 (CH1) and becomes part of this heavy chain. Meanwhile, the corresponding VH is fused to the light chain constant region (CL) and becomes a part of this light chain.
[0403] The present disclosure provides a different strategy. The reason of employing technologies, e.g., CrossMAb or common light chain, is because conventional antibodies have and need both heavy and light chains to function and / or maintain stability. However, target binding does not necessarily require both heavy and light chains. For example, a Fab fragment (Fab) contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain, is fully functional for antigen-binding. The variable domain of heavy chain antibodies (VHH) , e.g., derived from camelids such as llama, camel, or alpaca, is fully functional for antigen-binding. By fusing antibody fragments (e.g., Fab or VHH) to human Fc with heterodimer preference, multispecific antibodies or antigen-binding proteins can be generated without considering the mismatch between heavy and light chains.
[0404] In this disclosure, a Fab that can bind to human CD3 and a VHH that can bind to one or more tumor associated antigens (e.g., CAIX) is provided. Such a design gives rise to 8 different CD3 / CAIX bispecific antibody molecules (see Structures 2-1 to 2-8 in FIG. 2) , 12 different CD3 / CAIX / CAIX multispecific antibodies (see Structures 3-1 to 3-8 in FIG. 3 and Structures 4-1 to 4-4 in FIG. 4) .
[0405] Anti-CD3 antibodies or antigen binding fragments thereof
[0406] In some embodiments, the multispecific antigen-binding proteins or multispecific antibodies (MABs) comprise antibodies or antigen-binding fragments thereof that specifically bind to CD3. Provided herein are antibodies or antigen-binding fragments (e.g., Fab) thereof that specifically bind to the epsilon chain of CD3 (CD3ε, referred to herein throughout as CD3) . Exemplary CDR sequences of anti-CD3 antibodies or antigen binding fragments thereof are provided in Tables 1-6.
[0407] Table 1. CDR sequences of the heavy chain of anti-CD3 antibodies according to the Kabat definition
[0408] Table 2. CDR sequences of the light chain of anti-CD3 antibodies according to the Kabat definition
[0409] Table 3. CDR sequences of the heavy chain of anti-CD3 antibodies according to the Chothia definition
[0410] Table 4. CDR sequences of the light chain of anti-CD3 antibodies according to the Chothia definition
[0411] Table 5. CDR sequences of the heavy chain of anti-CD3 antibodies according to the IMGT definition
[0412] Table 6. CDR sequences of the light chain of anti-CD3 antibodies according to the IMGT definition
[0413] As provided herein, the anti-CD3 antibodies and antigen binding fragments thereof described herein comprise at least one set of the CDR 1, 2, and 3 sequences provided in Tables 1-6.
[0414] Exemplary VH and VL sequences of anti-CD3 antibodies and antigen binding fragments thereof are provided in Tables 7-8. As provided herein, the anti-CD3 antibodies or antigen binding fragments thereof described herein comprise at least one pair of the VH and VL sequences provided in Tables 7-8.
[0415] Table 7. VH sequences of anti-CD3 antibodies
[0416] Table 8. VL sequences of anti-CD3 antibodies
[0417] Table 9. CH1, CL, and linker sequences
[0418] The disclosure provides anti-CD3 antibodies, and antigen binding fragments (e.g., Fab) thereof, comprising a set of the CDR sequences in Tables 1-6, VH and VL pairs in Tables 7-8, and / or the CH1 and CL sequences in Table 9. In some embodiments, the antigen binding fragments described herein are anti-CD3 Fabs.
[0419] In some embodiments, the term “153-Ab” described herein may represent any one of the anti-CD3 Fabs.
[0420] Exemplary anti-CD3 Fabs comprise a VH (e.g., any of the VH described in Table 7) , a VL (e.g., any of the VL described in Table 8) , a CH1 (e.g., CH1 described in Table 9) and a CL (e.g., CL described in Table 9) .
[0421] Exemplary anti-CD3 Fabs comprise a first part (e.g., any of the first part of the Fab described in Table 10) comprising a VH (e.g., any of the VH described in Table 7) and a CH1 (e.g., CH1 described in Table 9) ; and a second part (e.g., any of the second part of the Fab described in Table 11) comprising a VL (e.g., any of the VL described in Table 8) and a CL (e.g., CL described in Table 9) .
[0422] Table 10 First chain (VH-CH1) sequences of anti-CD3 Fabs
[0423] Table 11 Second chain (VL-CL) sequences of anti-CD3 Fabs
[0424] Activatable anti-CD3 antibodies or antigen binding fragments thereof
[0425] In some embodiments, the multispecific antigen-binding proteins or multispecific antibodies (MABs) comprise activatable antibodies or antigen-binding fragments thereof that specifically bind to CD3. In some embodiments, any one of the anti-CD3 antibodies and antigen binding fragments thereof provided herein can be in an activatable antibody format.
[0426] As generally provided herein, the anti-CD3 antibodies, antigen binding fragments thereof, of the disclosure can comprise a masking peptide (e.g., M1 as described herein) . Accordingly, as used herein, the term “prodomain” refers to a polypeptide comprising a masking peptide (e.g., M1 as described herein) and a cleavable linker (e.g., L1 as described herein) . In certain embodiments, a prodomain comprises one of the following formulas (where the formula below represents an amino acid sequence in either a N-to C-terminal direction or a C-to N-terminal direction) : M1-L1-MAB.
[0427] In exemplary embodiments, a prodomain comprises a CD3 mask peptide M1 and a cleavable linker L1 that is cleavable by a matriptase or MMP. In some embodiments, provided herein are activatable multispecific antibodies (MABs) comprising a prodomain. Also provided herein are nucleotides encoding any of the prodomains described herein.
[0428] In some embodiments, a masking moiety (M1) or a mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof comprises any one of the sequences set forth in Table 12.
[0429] Table 12 Masking moiety (M1) sequences
[0430] In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof may be screened using any of the anti-CD3 antibodies or antigen binding fragments thereof described herein. In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof can reduce or inhibit the binding of the anti-CD3 antibodies or antigen binding fragments thereof (e.g., any of the anti-CD3 Fabs described herein) to CD3. In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof can reduce or inhibit the binding of the anti-CD3 antibodies or antigen binding fragments thereof having the same heavy chain CDR sequences and / or the same light chain CDR sequences of any one of the anti-CD3 Fabs described herein. In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof can reduce or inhibit the binding of the anti-CD3 antibodies or antigen binding fragments thereof having the same heavy chain CDR sequences of any one of 153-Ab18, 153-Ab19, 153-Ab21～153-Ab28, 153-Ab33, and 153-Ab34. In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof can reduce or inhibit the binding of the anti-CD3 antibodies or antigen binding fragments thereof having the same light chain CDR sequences of any one of 153-Ab11, 153-Ab12, 153-Ab14, 153-Ab15, 153-Ab16, 153-Ab17, 153-Ab18, 153-Ab21, 153-Ab22, 153-Ab25, 153-Ab26, 153-Ab29, 153-Ab30, 153-Ab31, 153-Ab32, 153-Ab33, 153-Ab34, 153-Ab35, and 153-Ab36. In some embodiments, the masking moiety (M1) or the mask peptide of the anti-CD3 antibodies or antigen binding fragments thereof can reduce or inhibit the binding of the anti-CD3 antibodies or antigen binding fragments thereof having the same heavy chain CDR sequences and the same light chain CDR sequences of any one of 153-Ab18, 153-Ab21, 153-Ab22, 153-Ab25, 153-Ab26, 153-Ab33, and 153-Ab34.
[0431] In some embodiments, the cleavable linker described herein (e.g., L1) comprises any one of the sequences set forth in Table 13. In some embodiments, the cleavable linker (e.g., L1 ) of an activatable multispecific antibodies (MABs) of the disclosure comprises any one of the sequences set forth in Table 13.
[0432] Table 13 Cleavable linker (L1) sequences
[0433] Anti-CAIX antibodies or antigen binding fragments thereof
[0434] In some embodiments, the multispecific antigen-binding proteins or multispecific antibodies (MABs) comprise antibodies or antigen-binding fragments thereof that specifically bind to CAIX. In some embodiments, the anti-CAIX antibodies or antibody fragments thereof comprise a variable domain of a heavy chain antibody (VHH) . Exemplary VHH sequences of anti-CAIX antibodies and antigen binding fragments thereof are provided in Table 14. The corresponding CDR sequences are provided in Tables 15-17.
[0435] Table 14 VHH sequences of anti-CAIX antibodies
[0436] Table 15. CDR sequences of the VHH of anti-CAIX antibodies according to the Kabat definition
[0437] Table 16. CDR sequences of the VHH of anti-CAIX antibodies according to the chothia definition
[0438] Table 17. CDR sequences of the VHH of anti-CAIX antibodies according to the IMGT definition
[0439] As provided herein, the anti-CAIX antibodies described herein comprise at least one set of the CDR 1, 2, and 3 sequences provided in Tables 15-17.
[0440] Exemplary VHH sequences of anti-CAIX antibodies of the disclosure (variable domains) are provided in Table 14. In some embodiments, the CDRs of anti-CAIX antibodies provided in Table 14 are defined by IMGT, Kabat, Chothia, Contact, or AbM definition.
[0441] Cleavable linkers
[0442] Both the monospecific antibodies and the multispecific antibodies (MABs) of the disclosure may comprise at least one cleavable linker (provided in Table 13) , when masked and / or not activated. In some embodiments, the terms "cleavable linker" and "cleavable substrate" are interchangeable.
[0443] In some embodiments, the cleavable linker (e.g., L1 described herein) of monospecific antibodies or the multispecific antibodies (MABs) includes an amino acid sequence that can serve as a substrate for at least one protease, usually an extracellular protease. A cleavable linker (e.g., L1 described herein) can serve as a substrate for multiple proteases, e.g., a substrate for a serine protease and a second different protease, e.g., an MMP. In some embodiments, a cleavable linker (e.g., L1 described herein) can serve as a substrate for more than one serine protease, e.g., a matriptase and uPA (urokinase) . In some embodiments, a cleavable linker (e.g., L1 described herein) can serve as a substrate for more than one MMPs, e.g., MMP9 and MMP14. In the case of multispecific antibodies (MABs) , the cleavable linker (e.g., L1 described herein) may be selected based on a protease that is co-localized in a tissue with the desired target of at least one AB of the cleavable linker (e.g., L1 described herein) .
[0444] A variety of different conditions are known in which a target of interest is co-localized with a protease, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be a cancerous tissue, particularly the cancerous tissue of a solid tumor. There are reports in the literature of increased levels of proteases in a number of cancers, e.g., liquid tumors (e.g., leukemia, lymphoma, and myeloma) or solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90 (7) : 1414-1421. Non-limiting examples of diseases include: all types of cancers, (such as, but not limited to breast, lung, colorectal, gastric, glioblastoma, ovarian, endometrial, renal, sarcoma, skin cancer, cervical, liver, bladder, cholangiocarcinoma, prostate, melanomas, head and neck cancer (e.g., head and neck squamous cell cancer, pancreatic, etc. ) , rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, ischemia, etc. For example, indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL) , lymphoblastic diseases including multiple myeloma, and solid tumors, including lung, colorectal, prostate, pancreatic and breast, including triple negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including by way of non-limiting example, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as head and neck squamous cell cancer; esophageal cancer; lung cancer, such as by way of non-limiting example, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, such as by way of non-limiting example, renal cell carcinoma; and / or skin cancer, such as by way of non-limiting example, squamous cell cancer, basal cell carcinoma, or melanoma. In some embodiments, the cancer is a squamous cell cancer. In some embodiments, the cancer is a skin squamous cell carcinoma. In some embodiments, the cancer is an esophageal squamous cell carcinoma. In some embodiments, the cancer is a head and neck squamous cell carcinoma. In some embodiments, the cancer is a lung squamous cell carcinoma.
[0445] For specific cleavage by an enzyme, contact between the enzyme and the cleavable linker (e.g., L1) is made. When the monospecific antibodies or the multispecific antibodies (MABs) comprise at least a first AB coupled to a mask peptide and a cleavable linker, e.g., the monospecific antibodies comprises an AB coupled to a mask peptide via a cleavable linker, is in the presence of target and sufficient enzyme activity, the cleavable linker can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the cleavable linker and effect cleavage. It can readily be envisioned that an enzyme may be in the vicinity of the cleavable linker but is unable to cleave because of other cellular factors or protein modification of the enzyme.
[0446] Exemplary cleavable linkers of the disclosure are provided in Table 13 above. In some embodiments, the cleavable linker has a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, or a length in the range of 10-15 amino acids.
[0447] Mask peptides
[0448] In both the activatable monospecific CD3 monospecific antibodies, or the multispecific antibodies (MABs) described above, the monospecific antibodies or the multispecific antibodies (MABs) can contain a mask peptide. As described herein, the monospecific antibodies (AB) or the multispecific antibodies (MABs) of the disclosure can comprise a prodomain (e.g., any of the prodomains described herein) , which comprises a mask peptide.
[0449] In some embodiments, the mask peptide is selected for use with a specific antibody or antigen-binding fragment thereof (e.g., the anti-CD3 antibodies or antigen-binding fragments thereof) .
[0450] In some embodiments, the first mask peptide of anti-CD3 antibody is selected from Table 12. In certain embodiments, the mask peptide is not a natural binding partner of the AB. In some embodiments, the mask peptide contains no or substantially no homology to any natural binding partner of the AB. In other embodiments the mask peptide is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%similar (e.g., in terms of sequence identity or homology) to any natural binding partners of the AB. In some embodiments, the mask peptide is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%identical to any natural binding partners of the AB. In some embodiments, the mask peptide is no more than 50%identical to any natural binding partners of the AB. In some embodiments, the mask peptide is no more than 25%identical to any natural binding partners of the AB. In some embodiments, the mask peptide is no more than 20%identical to any natural binding partners of the AB. In some embodiments, the mask peptide is no more than 10%identical to any natural binding partners of the AB.
[0451] An example of a spacer joined directly to the N-terminus of the mask peptide of the activatable antibody is selected from the group consisting of QGQSGS (SEQ ID NO: 273) , GQSGS (SEQ ID NO: 274) ; QSGS (SEQ ID NO: 275) ; SGS (SEQ ID NO: 276) ; GS (SEQ ID NO: 277) ; S; QGQSGQG (SEQ ID NO: 278) ; GQSGQG (SEQ ID NO: 279) ; QSGQG (SEQ ID NO: 280) ; SGQG (SEQ ID NO: 281) ; GQG (SEQ ID NO: 282) ; QG (SEQ ID NO: 283) ; G; QGQSGQ (SEQ ID NO: 284) ; GQSGQ (SEQ ID NO: 285) ; QSGQ (SEQ ID NO: 286) ; SGQ (SEQ ID NO: 287) ; GQ (SEQ ID NO: 288) ; and Q.
[0452] Exemplary mask peptides of the disclosure can have a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, a length in the range of 10-15 amino acids, or a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
[0453] As provided herein, the mask peptide inhibits the binding of the AB to the target. The mask peptide binds to the antigen-binding domain of the AB and inhibits binding of the AB to the target. In some embodiments, the mask peptide can sterically inhibit the binding of the AB to the target. In some embodiments, the mask peptide can allosterically inhibit the binding of the AB to its target. In these embodiments when the AB is modified by or coupled to a mask peptide and in the presence of target, there is no binding or substantially no binding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%binding of the AB to the target, as compared to the binding of the AB not modified by or coupled to a mask peptide, the parental AB, or the AB not coupled to a mask peptide to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.
[0454] When an AB is coupled to or modified with a mask peptide, the mask peptide "masks" or reduces or otherwise inhibits the specific binding of the AB to the target. When an AB is coupled to or modified by a mask peptide, such coupling or modification can lead to a structural change that reduces or inhibits the ability of the AB to specifically bind to its target.
[0455] Anti-CD3 Antibodies and Antigen-Binding Fragments
[0456] The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD3. The antibodies and antigen-binding fragments described herein are capable of binding to CD3 and can promote CD3-associated signaling pathways.
[0457] The amino acid sequences for heavy chain variable regions and light variable regions are also provided. Any of the heavy chain variable region sequences (SEQ ID NOs: 62-80, 289) can be paired with any of the light chain variable region sequences (SEQ ID NO: 81-105, 290) . Any of the heavy chain variable region sequences shown in Table 7 can be paired with any of the light chain variable region sequences shown in Table 8. In some embodiments, the CDRs of anti-CD3 antibodies provided in Table 7 or Table 8 are defined by IMGT, Kabat, Chothia, Contact, or AbM definition.
[0458] In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in Tables 1-2 (Kabat CDR) , Tables 3-4 (Chothia CDR) and Table 5-6 (IMGT CDR) .
[0459] In some embodiments, the antibody or antigen-binding fragment described herein comprises VH CDRs 1, 2, 3, and VL CDRs 1, 2, 3. In some embodiments, the VH CDRs 1, 2, 3 are any VH CDRs 1, 2, 3 shown in Table 1 (Kabat CDR) , Table 3 (Chothia CDR) or Table 5 (IMGT CDR) . In some embodiments, the VL CDRs 1, 2, 3 are any VL CDRs 1, 2, 3 shown in Table 2 (Kabat CDR) , Table 4 (Chothia CDR) or Table 6 (IMGT CDR) . In some embodiments, the CDRs are defined by IMGT, Kabat, Chothia, Contact, or AbM definition.
[0460] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the VH CDR 1 with zero, one or two amino acid insertions, deletions, or substitutions; VH CDR 2 with zero, one or two amino acid insertions, deletions, or substitutions; VH CDR 3 with zero, one or two amino acid insertions, deletions, or substitutions. The VH CDRs 1, 2, 3 can be selected from Table 1, Table 3, or Table 5.
[0461] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the VL CDR 1 with zero, one or two amino acid insertions, deletions, or substitutions; VL CDR 2 with zero, one or two amino acid insertions, deletions, or substitutions; VL CDR3 with zero, one or two amino acid insertions, deletions, or substitutions. The VL CDRs 1, 2, 3 can be selected from Table 2, Table 4, and Table 6.
[0462] The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
[0463] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD3. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is any one of SEQ ID NOs: 62-80, and 289, and the selected VL sequence is any one of SEQ ID NOs: 81-105, and 290.
[0464] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0465] The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in Tables 1-6, or have sequences as shown in Tables 7-8. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to CD3 (e.g., human CD3) .
[0466] The anti-CD3 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
[0467] Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CD3 will retain an ability to bind to CD3. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
[0468] The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F (ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
[0469] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
[0470] Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) . Antibody homodimers can be converted to Fab’ 2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25: 396-404, 2002) .
[0471] In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.
[0472] Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
[0473] Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.
[0474] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
[0475] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .
[0476] Structures of multispecific antigen-binding proteins
[0477] The present disclosure provides an antigen-binding protein, comprising (a) a first antigen-binding domain and (b) a second antigen-binding domain. In some embodiments, the antigen-binding protein comprises a first mask peptide that impairs the binding of the first antigen-binding domain to the target of the first antigen-binding domain. In some embodiments, the antigen-binding protein further comprises a half-life extension unit.
[0478] In some embodiments, the first mask peptide (M1) is linked to the first antigen-binding domain through a cleavable linker.
[0479] In the antigen-binding protein, a cleavable linker may be used to couple a mask peptide to an antigen-binding domain. A cleavable linker can have a cleavable site and optionally two linkers, e.g., before and after the cleavable site. The cleavable linker can be any segments of amino acids conventionally used as a cleavable linker for joining peptide domains. Suitable cleavable likers can vary in length, such as 1-20, 2-15, 3-12, 4-10, 6-20, 6-10, 5, 6, 7, 8, 9 or 10 amino acids long. In some embodiments, the cleavable linker has at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the cleavable linker has no more than 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some embodiments, the cleavable linker has about 14～30 amino acids. In some embodiments, the cleavable linker has about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the cleavable site has about 6-10 amino acids. In some embodiments, the cleavable site in the cleavable linker has about 6, 7, 8, 9, or 10 amino acids.
[0480] A cleavable site may be recognized and cleaved by a protease expressed extracellularly so it contacts a masked antigen-binding construct (e.g., an antigen-binding domain) , releasing the masked antigen-binding construct and allowing it to contact its target, such as a receptor extracellular domain or soluble ligand. Several matrix metalloproteinase sites (MMP1-28) may be suitable. MMPs play a role in tissue remodeling and are implicated in neoplastic processes such as morphogenesis, angiogenesis and metastasis. Examples of MMPs are provided in US 2013 / 0309230, WO 2009 / 025846, WO 2010 / 081173, WO 2014 / 107599, WO 2015 / 048329, US 20160160263, and Ratnikov et al., Proc. Natl. Acad. Sci. USA, 111: E4148-E4155 (2014) ; each of which is incorporated herein by reference in its entirety.
[0481] The cleavable site may be cleaved in the tumor microenvironment. The cleavable site may be a matrix metalloprotease (MMP) cleavage site. The MMP cleavage site may be selected from an MMP2 cleavage site, an MMP7 cleavage site, an MMP9 cleavage site and an MMP13 cleavage site. Following cleavage by an MMP, the heavy chain and / or light chain of the masked antigen-binding construct may comprise a stub amino acid remnant of the MMP cleavage site. The stub amino acid remnant may contain the sequence LRSG, SG, VR, R, or K at the N terminus of the antigen-binding portion of the masked antigen-binding construct.
[0482] A cleavable site may also be cleaved at dibasic sites (e.g., an Arginine-Arginine, Lysine-Arginine, or Lysine-Lysine site) . Enzymes that cleave at dibasic sites are known in the art and include, for example, N-arginine dibasic convertase (Chow et al., JBC 275: 19545-19551 (2000) ) and subtilisin-like proprotein convertases such as Furin (PCI) , PC2, and PC3 (Steiner (1991) in Peptide Biosynthesis and Processing (Fricker ed. ) pp. 1-16, CRC Press, Boca Raton, FL; Muller et al., JBC 275: 39213-39222, (2000) ) .
[0483] In addition, one or more linkers can be used to couple a mask peptide to an antigen-binding domain. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral linker between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992) ) . Some exemplary linkers are in the form S (G) nS, wherein n is from 5-20. Other exemplary linkers are (G)n, glycine-serine polymers (including, for example, (GS) n, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
[0484] The multispecific antigen-binding proteins can be designed to include one or more antigen-binding domains that target T cell antigens (e.g., CD3) , and include one or more antigen-binding domains that target a tumor-associated antigen (e.g., CAIX) . The antigen-binding domain can comprise, e.g., a Fab, a scFv, a VHH-VHH, or a VHH. In some embodiments, the one or more antigen-binding domains that target a T cell antigen (e.g., CD3) can comprise a Fab. In some embodiments, the one or more antigen-binding domains that target the tumor-associated antigen can comprise a VHH. The tumor-associated antigen refers to an antigen that is specifically expressed on tumor cell surfaces. These antigens can be used to identify tumor cells. Normal cells rarely express these tumor associated antigens. Some exemplary tumor-associated antigens include, e.g., CAIX.
[0485] The present disclosure provides antigen-binding proteins with various formats as described herein. While not intending to be bound by any theory, it is hypothesized that the in the presence of the target cells (e.g., cancer cells) and T cells, the antigen-binding proteins can effectively activate T cells.
[0486] The multispecific antigen-binding proteins can be used to treat tumor associated antigen (TAA) -positive cancers in a subject (e.g., a human patient) . In some embodiments, the tumor associated antigen positive cancer is CAIX-positive cancer.
[0487] The multispecific antigen-binding proteins described herein may or may not contain a Fc region. In order to extend the serum half-life of the multispecific antigen-binding proteins, the multispecific antigen-binding proteins can contain one or more half-life extending units (E) . In some embodiments, the half-life extending unit can be a VHH that specifically binds to human serum albumin (HSA) . The amino acid sequence of the VHH is set forth in SEQ ID NO: 268 (EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS) .
[0488] In some embodiments, the half-life extending unit is located between a mask peptide and the antigen-binding domain. In some embodiments, the half-life extending unit is located between a mask peptide and a cleavable linker for the mask peptide. In some embodiments, the half-life extending unit is linked to N-terminus of the antigen-binding domain. In some embodiments, the half-life extending unit is linked to C-terminus of the antigen-binding domain. In some embodiments, the half-life extending unit is linked to C-terminus of the antigen-binding domain through a cleavable linker. In some embodiments, the half-life extending unit is linked to N-terminus of the mask peptide.
[0489] The multispecific antigen-binding proteins with various structures are described below.
[0490] B1 (Fab) -B2 (VHH)
[0491] As shown in Structure 1-1 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first chain of a Fab (B1) , and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0492] As shown in Structure 1-2 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first chain of a Fab (B1) , a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0493] As shown in Structure 1-11 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first chain of a Fab (B1) , and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) and a second VHH of the second antigen-binding domain (B2) .
[0494] In some embodiments, the first chain of a Fab (B1) and the second chain of the Fab (B1) can associate with each other, forming a Fab (B1) that specifically binds to an antigen (e.g., CD3) . In some embodiments, the second antigen-binding domain (B2) comprises one or more VHHs that specifically bind to an antigen (e.g., CAIX) . In some embodiments, the first chain of a Fab (B1) comprises any of the amino acid sequences in Table 10. In some embodiments, the second chain of a Fab (B1) comprises any of the amino acid sequences in Table 11. In some embodiments, the second antigen-binding domain (B2) comprises any of the amino acid sequences in Table 14.
[0495] In some embodiments, the multispecific antigen-binding protein comprises a masking moiety (M1) that impairs the binding of B1 with its target. In some embodiments, M1 comprises any one of the amino acid sequences shown in Table 12.
[0496] As shown in Structure 1-5 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0497] As shown in Structure 1-6 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0498] As shown in Structure 1-7 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first mask peptide (M1) , a cleavable linker, and a first chain of a Fab (B1) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) , and a VHH of a second antigen-binding domain (B2) .
[0499] As shown in Structure 1-8 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first mask peptide (M1) , a cleavable linker, and a first chain of a Fab (B1) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) , a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) .
[0500] As shown in Structure 1-12 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) and a second VHH of the second antigen-binding domain (B2) .
[0501] In some embodiments, the multispecific antigen-binding protein further comprises a half-life extension unit (E) .
[0502] As shown in Structure 1-3 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0503] As shown in Structure 1-4 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) .
[0504] As shown in Structure 1-9 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, and a first chain of a Fab (B1) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) , and a VHH of a second antigen-binding domain (B2) .
[0505] As shown in Structure 1-10 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, and a first chain of a Fab (B1) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) , a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) .
[0506] As shown in Structure 1-13 in FIG. 1, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a first chain of a Fab (B1) , and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a second chain of the Fab (B1) and a second VHH of the second antigen-binding domain (B2) .
[0507] In some embodiments, the first half-life extending unit (E1) comprises a VHH that specifically binds to human serum albumin (HSA) .
[0508] In some embodiments, the first chain of a Fab (B1) and the second chain of the Fab (B1) can associate with each other, forming a Fab (B1) that specifically binds to an antigen (e.g., CD3) . In some embodiments, the second antigen-binding domain (B2) comprises one or more VHHs that specifically bind to an antigen (e.g., CAIX) .
[0509] In some embodiments, the first chain of a Fab comprises a heavy chain variable region (VH) and a CH1 domain and the second chain of the Fab comprises a light chain variable region (VL) and a CL domain. In some embodiments, the first chain of a Fab comprises a VL and a CL domain and the second chain of the Fab comprises a VH and a CH1 domain. In some embodiments, the first chain of a Fab comprises a VH and a CL domain and the second chain of the Fab comprises a VL and a CH1 domain. In some embodiments, the first chain of a Fab comprises a VL and a CH1 domain and the second chain of the Fab comprises a VH and a CL domain. Schematic structures of exemplary multispecific antigen-binding protein are shown in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.
[0510] As shown in Structure 2-1 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CL domain, and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL and a CH1 domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0511] As shown in Structure 2-2 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CL and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH and a CH1 domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0512] As shown in Structure 2-3 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CH1 domain, and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0513] As shown in Structure 2-4 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CH1 domain, and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0514] As shown in Structure 2-5 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, and a CL domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CH1 domain, and a VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0515] As shown in Structure 2-6 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, and a CL domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain, and a VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0516] As shown in Structure 2-7 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, and a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, and a VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0517] As shown in Structure 2-8 in FIG. 2, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, and a CH1 domain; (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH, a CL domain, and a VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0518] As shown in Structure 3-1 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CL domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL and a CH1 domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0519] As shown in Structure 3-2 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CL domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH and a CH1 domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0520] As shown in Structure 3-3 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CH1 domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0521] As shown in Structure 3-4 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CH1 domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH and a CL domain. In some embodiments, the VH and the VL associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0522] As shown in Structure 3-5 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, and a CL domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CH1 domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0523] As shown in Structure 3-6 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL and a CL domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0524] As shown in Structure 3-7 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, and a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0525] As shown in Structure 3-8 in FIG. 3, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, and a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH , a CL domain, a first VHH of a second antigen-binding domain (B2) and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0526] As shown in Structure 4-1 in FIG. 4, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CL domain, and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CH1 domain, and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0527] As shown in Structure 4-2 in FIG. 4, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CL domain, and a first VHH of a second antigen-binding domain (B2) ; (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain, and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0528] As shown in Structure 4-3 in FIG. 4, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VH, a CH1 domain, a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0529] As shown in Structure 4-4 in FIG. 4, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a half-life extension unit (E) , a first mask peptide (M1) , a cleavable linker, a VL, a CH1 domain, and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VH, a CL domain, and a second VHH of the second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the first and second VHHs of the second antigen-binding domain (B2) specifically bind to a tumor-associated antigen (e.g., CAIX) .
[0530] As shown in Structure 5-1A in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, and a VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0531] As shown in Structure 5-2B in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VHH of a second antigen-binding domain (B2) , a VL and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0532] As shown in Structure 5-3C in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, and a first VHH of a second antigen-binding domain (B2) , and a second VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0533] As shown in Structure 5-4D in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a first VHH of a second antigen-binding domain (B2) , a second VHH of a second antigen-binding domain (B2) , a VL and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0534] As shown in Structure 5-5E in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, a CH1 domain, and a first VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain, and a second VHH of a second antigen-binding domain (B2) . In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0535] As shown in Structure 5-6F in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, and a CH1 domain, and a VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0536] As shown in Structure 5-7G in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VH, and a CH1 domain, a first VHH of a second antigen-binding domain (B2) , and a second VHH of a second antigen-binding domain (B2) ; and (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0537] As shown in Structure 5-8H in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a VHH of a second antigen-binding domain (B2) , a VH, and a CH1 domain; (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0538] As shown in Structure 5-9I in FIG. 5, a multispecific antigen-binding protein can be prepared, which includes: (a) a first polypeptide including, preferably from N-terminus to C-terminus: a first VHH of a second antigen-binding domain (B2) , a second VHH of a second antigen-binding domain (B2) a VH, and a CH1 domain; (b) a second polypeptide including, preferably from N-terminus to C-terminus: a VL, and a CL domain. In some embodiments, the VH and the VL can associate with each other, forming an antigen-binding site of a Fab (B1) that specifically binds to a T cell antigen (e.g., CD3) . In some embodiments, the VHH of the second antigen-binding domain (B2) specifically binds to a tumor-associated antigen (e.g., CAIX) .
[0539] In some embodiments, the first and second VHHs described herein are the same. In some embodiments, the first and second VHHs described herein are different. As used herein, the first and second VHHs are not necessarily within the same protein domain. When the first and / or second VHHs are referred to as “of the second antigen-binding domain (B2) , ” it indicates that the first and / or second VHHs can specifically bind to the target of the second antigen-binding domain (B2) , e.g., CAIX.
[0540] In one aspect, provided herein is a multispecific antigen-binding protein comprising an anti-CD3 Fab (e.g., any of the anti-CD3 Fabs described herein) , an anti-CAIX VHH (e.g., any of the anti-CAIX VHHs described herein) , and an anti-CD3 mask (e.g., any of the anti-CD3 masks described herein, including the masking moieties in Table 12) . In some embodiments, the anti-CD3 Fab comprises a VH selected from Table 7, a VL selected from Table 8, a CH1 domain and a CL domain in Table 9. In some embodiments, the anti-CAIX VHH is selected from Table 14. In some embodiments, the anti-CD3 mask is selected from Table 12.
[0541] In one aspect, provided herein is a multispecific antigen-binding protein comprising an anti-CD3 Fab, an anti-CAIX VHH and an anti-CD3 mask shown in Table 18. In some embodiments, the term “153-Ab” represents any one of the anti-CD3 Fabs described herein, including the anti-CD3 Fabs (from 153-Ab1 to 153-Ab36) in Tables 10-11. In some embodiments, the term “193-Ab” represents any one of the anti-CAIX VHHs described herein, including the anti-CAIX VHHs (from 193-Ab1 to 193-Ab20) in Table 14. In some embodiments, the term “ma-153” represents any one of the anti-CD3 masks described herein, including the anti-CD3 masks (from ma-153-1 to ma-153-17) in Table 12.
[0542] For example, for MAB, the anti-CD3 Fab can be 153-Ab (e.g., 153-Ab1) , the anti-CAIX VHH can be 193-Ab (e.g., 193-Ab1) , and the anti-CD3 mask can be mask-153 (e.g., ma-153-1) . In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0543] For example, for MAB1, the anti-CD3 Fab can be 153-Ab1 , the anti-CAIX VHH can be 193-Ab1, and the anti-CD3 mask can be ma-153-1. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0544] For example, for MAB2, the anti-CD3 Fab can be 153-Ab2 , the anti-CAIX VHH can be 193-Ab2, and the anti-CD3 mask can be ma-153-2. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0545] For example, for MAB3, the anti-CD3 Fab can be 153-Ab3 , the anti-CAIX VHH can be 193-Ab3, and the anti-CD3 mask can be ma-153-3. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0546] For example, for MAB4, the anti-CD3 Fab can be 153-Ab4 , the anti-CAIX VHH can be 193-Ab4, and the anti-CD3 mask can be ma-153-4. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0547] For example, for MAB5, the anti-CD3 Fab can be 153-Ab8 , the anti-CAIX VHH can be 193-Ab5, and the anti-CD3 mask can be ma-153-5. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0548] For example, for MAB6, the anti-CD3 Fab can be 153-Ab11 , the anti-CAIX VHH can be 193-Ab6, and the anti-CD3 mask can be ma-153-6. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0549] For example, for MAB7, the anti-CD3 Fab can be 153-Ab14 , the anti-CAIX VHH can be 193-Ab7, and the anti-CD3 mask can be ma-153-7. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0550] For example, for MAB8, the anti-CD3 Fab can be 153-Ab15 , the anti-CAIX VHH can be 193-Ab8, and the anti-CD3 mask can be ma-153-8. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0551] For example, for MAB9, the anti-CD3 Fab can be 153-Ab16 , the anti-CAIX VHH can be 193-Ab9, and the anti-CD3 mask can be ma-153-9. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0552] For example, for MAB10, the anti-CD3 Fab can be 153-Ab18 , the anti-CAIX VHH can be 193-Ab10, and the anti-CD3 mask can be ma-153-10. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0553] For example, for MAB11, the anti-CD3 Fab can be 153-Ab32 , the anti-CAIX VHH can be 193-Ab11, and the anti-CD3 mask can be ma-153-1. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0554] For example, for MAB12, the anti-CD3 Fab can be 153-Ab36 , the anti-CAIX VHH can be 193-Ab12, and the anti-CD3 mask can be ma-153-2. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0555] For example, for MAB13, the anti-CD3 Fab can be 153-Ab1 , the anti-CAIX VHH can be 193-Ab13, and the anti-CD3 mask can be ma-153-3. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0556] For example, for MAB14, the anti-CD3 Fab can be 153-Ab2 , the anti-CAIX VHH can be 193-Ab14, and the anti-CD3 mask can be ma-153-4. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0557] For example, for MAB15, the anti-CD3 Fab can be 153-Ab3 , the anti-CAIX VHH can be 193-Ab15, and the anti-CD3 mask can be ma-153-5. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0558] For example, for MAB16, the anti-CD3 Fab can be 153-Ab4, the anti-CAIX VHH can be 193-Ab16, and the anti-CD3 mask can be ma-153-6. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0559] For example, for MAB17, the anti-CD3 Fab can be 153-Ab8, the anti-CAIX VHH can be 193-Ab17, and the anti-CD3 mask can be ma-153-7. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0560] For example, for MAB18, the anti-CD3 Fab can be 153-Ab18, the anti-CAIX VHH can be 193-Ab18, and the anti-CD3 mask can be ma-153-8. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0561] For example, for MAB19, the anti-CD3 Fab can be 153-Ab14, the anti-CAIX VHH can be 193-Ab19, and the anti-CD3 mask can be ma-153-9. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0562] For example, for MAB20, the anti-CD3 Fab can be 153-Ab15, the anti-CAIX VHH can be 193-Ab20, and the anti-CD3 mask can be ma-153-10. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0563] Table 18
[0564] In one aspect, provided herein is a multispecific antigen-binding protein comprising an anti-CD3 Fab, and an anti-CAIX VHH shown in Table 19. In some embodiments, the term “153-Ab” represents any one of the anti-CD3 Fabs described herein, including the anti-CD3 Fabs (from 153-Ab1 to 153-Ab36) in Tables 10-11. In some embodiments, the term “193-Ab” represents any one of the anti-CAIX VHHs described herein, including the anti-CAIX VHHs (from 193-Ab1 to 193-Ab20) in Table 14.
[0565] For example, for MAB, the anti-CD3 Fab can be 153-Ab (e.g., 153-Ab1 to153-Ab36) , and the anti-CAIX VHH can be 193-Ab (e.g., 193-Ab1 to 193-Ab20) . In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0566] For example, for MAB21, the anti-CD3 Fab can be 153-Ab1 , and the anti-CAIX VHH can be 193-Ab1. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0567] For example, for MAB22, the anti-CD3 Fab can be 153-Ab2 , and the anti-CAIX VHH can be 193-Ab2. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0568] For example, for MAB23, the anti-CD3 Fab can be 153-Ab3 , and the anti-CAIX VHH can be 193-Ab3. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0569] For example, for MAB24, the anti-CD3 Fab can be 153-Ab4 , and the anti-CAIX VHH can be 193-Ab4. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0570] For example, for MAB25, the anti-CD3 Fab can be 153-Ab8 , and the anti-CAIX VHH can be 193-Ab5. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0571] For example, for MAB26, the anti-CD3 Fab can be 153-Ab11 , and the anti-CAIX VHH can be 193-Ab6. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0572] For example, for MAB27, the anti-CD3 Fab can be 153-Ab14 , and the anti-CAIX VHH can be 193-Ab7. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0573] For example, for MAB28, the anti-CD3 Fab can be 153-Ab15 , and the anti-CAIX VHH can be 193-Ab8. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0574] For example, for MAB29, the anti-CD3 Fab can be 153-Ab16 , and the anti-CAIX VHH can be 193-Ab9. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0575] For example, for MAB30, the anti-CD3 Fab can be 153-Ab18, and the anti-CAIX VHH can be 193-Ab10. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0576] For example, for MAB31, the anti-CD3 Fab can be 153-Ab32 , and the anti-CAIX VHH can be 193-Ab11. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0577] For example, for MAB32, the anti-CD3 Fab can be 153-Ab36 , and the anti-CAIX VHH can be 193-Ab12. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0578] For example, for MAB33, the anti-CD3 Fab can be 153-Ab1 , and the anti-CAIX VHH can be 193-Ab13. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0579] For example, for MAB34, the anti-CD3 Fab can be 153-Ab2 , and anti-CAIX VHH can be 193-Ab14. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0580] For example, for MAB35, the anti-CD3 Fab can be 153-Ab3 , and the anti-CAIX VHH can be 193-Ab15. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0581] For example, for MAB36, the anti-CD3 Fab can be 153-Ab4, and the anti-CAIX VHH can be 193-Ab16. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0582] For example, for MAB37, the anti-CD3 Fab can be 153-Ab8, and the anti-CAIX VHH can be 193-Ab17. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0583] For example, for MAB38, the anti-CD3 Fab can be 153-Ab18, and the anti-CAIX VHH can be 193-Ab18 In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0584] For example, for MAB39, the anti-CD3 Fab can be 153-Ab14, and the anti-CAIX VHH can be 193-Ab19. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0585] For example, for MAB40, the anti-CD3 Fab can be 153-Ab15, and the anti-CAIX VHH can be 193-Ab20. In some embodiments, the structure of the multispecific antigen-binding protein is selected from any of the schematic structures described herein, particularly, Structure 5-7G in FIG. 5.
[0586] Table 19
[0587] In some embodiments, any particular combination of the anti-CD3 Fab, the anti-CAIX VHH and the anti-CD3 mask shown in Table 18 can have a structure of the multispecific antigen-binding protein that is selected from any of the schematic structures described herein, particularly, Structure 1-4, Structure 1-8, or Structure 1-12 in FIG. 1.
[0588] In some embodiments, any particular combination of the anti-CD3 Fab, and the anti-CAIX VHH shown in Table 19 can have a structure of the multispecific antigen-binding protein that is selected from any of the schematic structures described herein, particularly, Structure 5-3C, or Structure 5-7G in FIG. 5
[0589] In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises VH CDRs 1, 2, 3, and / or VL CDRs 1, 2, 3. In some embodiments, the VH CDRs 1, 2, 3 are the same as the antibodies shown in Table 18 or Table 19. In some embodiments, the VL CDRs 1, 2, 3 are the same as the antibodies shown in Table 18 or Table 19. In some embodiments, the CDRs are defined by IMGT, Kabat, Chothia, Contact, or AbM definition.
[0590] Methods of Making Antibodies, Antigen-Binding Fragments, or Antigen-Binding Proteins
[0591] An isolated fragment of a human protein (e.g., CD3 or CAIX) can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .
[0592] The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
[0593] An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
[0594] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of the protein) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds. ) , John Wiley & Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
[0595] VHH can also be obtained from or designed synthetic llama VHH libraries. PBMC from llamas can be obtained, and RNA can be isolated to generate cDNA by reverse transcription. Then, the VHH genes can be amplified by PCR and cloned to a phage display vector to construct the VHH library. The synthetic (e.g., humanized) VHH library can be prepared by incorporation of shuffled VHH CDR1, 2 and 3, generated by overlapping PCR, to a modified human VH scaffold to generate enhanced diversity and keep low immunogenicity. The VHH libraries can be then panned against antigens to obtain VHH with desired binding affinities.
[0596] Variants of the antibodies, antigen-binding fragments, or the antigen-binding proteins described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.
[0597] Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.
[0598] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.
[0599] A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ; each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.
[0600] It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.
[0601] Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
[0602] In some embodiments, a covalent modification can be made to the antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.
[0603] In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
[0604] In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al. “The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. ” Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
[0605] In some embodiments, the methods described here are designed to make a bispecific antibody. In some embodiments, the methods described here are designed to make a trispecific antibody. Bispecific or trispecific antibodies can be made by engineering the interface between different antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.
[0606] In some embodiments, one or more amino acid residues in the CH3 portion of the IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions T366W. The other heavy chain can have one or more the following substitutions T366S, L368A, and Y407V. Furthermore, a substitution (-ppcpScp-->-ppcpPcp-) can also be introduced at the hinge regions of both substituted IgG.
[0607] Furthermore, an anion-exchange chromatography can be used to purify the antibodies or antigen-binding fragments described herein. Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE) . In solution, the resin is coated with positively charged counter-ions (cations) . Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography can be used to purify proteins based on their isoelectric point (pI) . The isoelectric point is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. Thus, in some embodiments, different amino acid substitution can be introduced into two heavy chains, so that the pI for the homodimer comprising two Arm A and the pI for the homodimer comprising two Arm B is different. The pI for the bispecific or trispecific antibody having Arm A and Arm B will be somewhere between the two pIs of the homodimers. Thus, the two homodimers and the bispecific antibody or trispecific antibody can be released at different pH conditions. The present disclosure shows that a few amino acid residue substitutions can be introduced to the heavy chains to adjust pI.
[0608] Methods of Treatment
[0609] The methods described herein include methods for the treatment of disorders associated with cancer. Generally, the methods include administering a therapeutically effective amount of multispecific antigen-binding proteins as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
[0610] As used in this context, to “treat” means to ameliorate at least one symptom of the disorder associated with cancer. Often, cancer results in death; thus, a treatment can result in an increased life expectancy (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years) . Administration of a therapeutically effective amount of an agent described herein (e.g., antigen-binding proteins) for the treatment of a condition associated with cancer will result in decreased number of cancer cells and / or alleviated symptoms.
[0611] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
[0612] In one aspect, the disclosure also provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and / or duration of one or more symptoms of the cancer in a subject.
[0613] In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, antigen-binding proteins, or an antibody drug conjugate disclosed herein to a subject in need thereof, e.g., a subject having, or identified or diagnosed as having, a cancer, e.g., breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.
[0614] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
[0615] In some embodiments, the cancer is a cancer expressing CAIX.
[0616] In some embodiments, the cancers are KIRC, CHOL, PAAD, LUSC, LUAD, HNSC, BLCA, ESCA, COAD, READ, SCLC, STAD, MESO and CESC.
[0617] In some embodiments, the cancer cells have an elevated CAIX level, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%higher than non-cancerous cells. In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.
[0618] As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen-binding fragment, antigen-binding protein c, antibody-drug conjugates, antibody-encoding polynucleotide, vector comprising the polynucleotide, and / or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
[0619] An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody, an antigen-binding fragment, an antigen-binding protein, or an antibody-drug conjugate is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and / or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective amount may vary, depending on, inter alia, patient history as well as other factors such as the type (and / or dosage) of the agent used.
[0620] Effective amounts and schedules for administering the antibodies, antigen-binding proteins, antibody-encoding polynucleotides, antibody-drug conjugates, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art.
[0621] A typical dosage of an effective amount of an antibody or antigen-binding protein is 0.01 μg / kg to 500 mg / kg. In some embodiments, the dosage is 0.01 μg / kg to 10 mg / kg. In some embodiments, the dosage is 0.1 μg / kg to 1 mg / kg. In some embodiments, the dosage can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.
[0622] In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, antigen-binding proteins, antibody-drug conjugates, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding proteins, antigen-binding protein constructs, antibody-drug conjugates, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
[0623] Pharmaceutical Compositions and Routes of Administration
[0624] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody, antigen-binding fragment thereof, or the antigen-binding protein can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .
[0625] Compositions containing one or more of any of the antibodies, antigen-binding proteins, antibody-drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
[0626] Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
[0627] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies, antigen-binding proteins, or antigen-binding protein constructs (e.g., any of the antibodies, antibody fragments, or antigen-binding proteins described herein) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and / or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antibodies, antigen-binding proteins, or antigen-binding protein constructs described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases) .
[0628] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding proteins, antigen-binding protein constructs, or antibody-drug conjugates described herein per kilogram of the subject’s weight (e.g., about 0.01 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding proteins, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody, antibody fragment, or antigen-binding proteins in vivo.
[0629] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies, antigen-binding proteins, or antigen-binding protein constructs for various uses as described herein.
[0630] EXAMPLES
[0631] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
[0632] Unless otherwise stated, temperatures are in degrees Celsius, and pressures are atmospheric or near atmospheric. Standard abbreviations may be used, such as bp for base pairs; kb for kilobase pairs; pL for picoliters; s or sec for seconds; min for minutes; h or hr for hours; aa for amino acids; nt for nucleotides; i. m. for intramuscular, etc.
[0633] Materials and Methods
[0634] DNA Sequencing and synthesis
[0635] The DNA sequence was determined by double-stranded DNA sequencing, and the sequencing was performed by GENEWIZ (Suzhou Jinweizhi Biotechnology Co., Ltd. ) .
[0636] Isolation of human Peripheral Blood Mononuclear Cells (PBMCs) .
[0637] Density gradient centrifugation was used to prepare PBMCs. Blood samples were obtained from a blood bank or healthy donors. The blood samples were stored in EDTA-containing anticoagulant tubes for 10 minutes. An equal volume of PBS solution containing 2%FBS (pH 7.4) was added and mixed well. 15 mL of Ficoll Paque PLUS density gradient medium (GE) was added to a 50 mL Falcon tube, and 30 mL of diluted fresh blood from the anticoagulant tube was gently added (along the tube wall) onto the density gradient medium in the Falcon tube. The centrifuge was set to brake-off mode. The tube was gently placed in the centrifuge, and centrifuged at 1450 rpm at room temperature for 45 minutes. The tube was carefully removed. The tube contained three layers: the upper serum layer, the middle white layer of PBMCs, and the bottom red blood cell layer. The upper yellow transparent serum layer was discarded and the middle layer was carefully transferred to a new 15 mL centrifuge tube. An equal volume of gradient medium was added and the tube was centrifuged at 1450 rpm at room temperature for 30 minutes. The upper supernatant was discarded and the cell pellet at the bottom was collected. The cells were resuspended and washed three times until the supernatant is clear. 1 mL of 2%FBS-PBS buffer was used to resuspend the cells. After counting the PBMCs, the cells were resuspended in RPMI 1640 medium containing 10%FBS (GIBCO) and 1%L-glutamine (GIBCO) , and cultured in a CO2 incubator at 37℃ with 5%CO2.
[0638] Example 1: Preparation of CD3 / CAIX Bispecific Antibodies with Different Structures
[0639] Based on the murine CD3 antibody SP-34, the humanized CD3 antibody TA1 was obtained through mutation, library construction, humanization, and multiple screenings. The CAIX single-domain antibody is referred to as anti-CAIX VHH in the structural diagram in FIG. 5.
[0640] According to FIG. 5, the structures of the bispecific antibodies CAIX-BsAb based on TA1-Fab (monovalent for CD3) and CAIX VHH (monovalent or bivalent for CAIX) were designed. In FIG. 5, “TA1 Fab” represents any one of the anti-CD3 Fabs described herein, including the anti-CD3 Fabs (from 153-Ab1 to 153-Ab36) in Tables 10-11; “CA IX VHH” represents any one of the anti-CAIX VHHs described herein, including the anti-CAIX VHHs (from 193-Ab1 to 193-Ab20) in Table 14.
[0641] In the TA1-Fab, TA1-Fab-VH was located at the N-terminus of CH1, and TA1-Fab-VL was located at the N-terminus of CL. The single-domain antibody CAIX VHH is located at either the N-or C-terminus of TA1-Fab. In this embodiment, TA1F represents the Fab region of the anti-CD3 antibody clone TA1, where TA1 VH is connected to CH1 (IgG4) , and TA1 VL is connected to CL (kappa) . The light chain is a common kappa light chain, and CH1 is derived from IgG4. For example, as shown in FIG. 5-7G, in one embodiment, the heavy chain of the CD3 / CAIX bispecific antibody is: CD3-Fab-VH-CH1 + anti-CAIX VHH + anti-CAIX VHH; the light chain is: CD3-Fab-VL-CL. In this embodiment, TA1F represents the Fab region of the anti-CD3 antibody clone TA1, where TA1 VH is connected to CH1 (IgG4) , and TA1 VL is connected to CL (kappa) . The light chain is a common kappa light chain, and CH1 is derived from IgG4.
[0642] In some embodiments, the bispecific antibodies adopt the above-mentioned design based on the CD3-Fab framework (FIG. 5) . Plasmids were constructed and co-transfected into 293F cells via PEI-mediated transfection. After protein expression and purification, CD3 / CAIX bispecific antibodies with a purity of ≥90%were obtained.
[0643] Example 2: CD3 / CAIX Bispecific Antibodies Induce T Cell-Mediated Killing of CAIX-Positive Tumor Target Cells
[0644] The functional activity of the bispecific antibodies were evaluated through tumor cell line killing assays. The T cell-mediated killing on target cells induced by the bispecific antibodies were evaluated. Human colorectal cancer cells HT-29 (moderate CAIX expression) were used as the target cells. Human PBMCs were used as effector cells. The killing was measured after 72 hours of incubation with the antibodies.
[0645] The tumor cells were digested with trypsin / EDTA, washed once with pre-cooled PBS, resuspended in RPMI 1640 medium containing 10%FBS, and seeded at a density of 5,000 cells / well in a flat-bottom 96-well plate (Corning 3599) . After incubating for 4 hours, 50 μL of serially diluted antibody solutions were added, with three replicates for each concentration. The antibodies and the cells were incubated for more than 30 minutes to allow the antibodies to fully bind to the cells. PBMCs were thawed, and added to 10 mL of RPMI 1640 medium containing 10%FBS, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended thoroughly. The cell density was adjusted according to experimental needs. PBMCs were added at 50 μL per well to the target cells to reach a final E: T ratio of 0.33: 1. The 96-well plate was incubated at 37℃ in a 5%CO2 incubator for 3 days. Finally, the CCK8 method (Dojindo, CK04-20) was used to detect cell viability. The cell survival rate was determined using GraphPad Prism software. The experimental results were shown as mean ± standard deviation (Mean±SD) or killing EC50 (nM) .
[0646] The experimental results are shown in the table below. The CD3 / CAIX bispecific antibodies have significant killing effects on tumor cells, with the strength of killing in the order of 5-7G > 5-3C > 5-5E > 5-4D > 5-9I > 5-1A > 5-8H > 5-6F > 5-2B. The same killing strength ranking was observed using different anti-CD3 Fabs and anti-CAIX VHHs, indicating that the 5-7G structure provides the best killing strength, regardless of the specific anti-CD3 Fab and anti-CAIX VHH.
[0647] Table 20: Killing Effect of CD3 / CAIX Bispecific Antibodies on HT-29 Tumor Cells (EC50, nM)
[0648] Example 3: In Vivo Antitumor Activity of CD3 / CAIX Bispecific Antibodies in OSRC2 Human Kidney Cancer Cell Xenografts
[0649] 3x106 OSRC2 cells were subcutaneously injected into female NCG (NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt, GemPharmatech, Strain NO. T001475) mice (n=6) with an injection volume of 200 μL per mouse. Three days later, 4x106 human PBMCs were subcutaneously injected into each mouse (E: T ratio of 4: 3) . To evaluate the treatment effects of the control bispecific antibodies, when the tumors reached 120-150mm3, different doses of the bispecific antibodies (0.03 mg / kg and 0.01 mg / kg) were intravenously injected once per day. The vehicle control group received PBS buffer instead of antibodies. A total of 7 doses were administered. The mice's body volume was measured and recorded every two days after administration.
[0650] The tumor volume was calculated based on the formula 1 / 2 ×long diameter × short diameter2. The tumor growth inhibition rate was calculated based on the formula TGITV (%) (tumor growth inhibition %) = [1 - (Ti -T0) / (Vi -V0) ] × 100%; Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group at the first measurable time point; Vi: the average tumor volume of the vehicle control group on the i-th day of administration, V0: the average tumor volume of the vehicle control group at the first measurable time point.
[0651] The experimental results were expressed as mean ± standard error (Mean±SEM) . Graphs were plotted using GraphPad Prism 9.0 software, and two-way ANOVA was performed. A P-value < 0.05 indicates that the differences are statistically significant.
[0652] The results are shown in FIG. 6 and the table below. On the 19th day of administration, compared to the vehicle control group, the CD3 / CAIX bispecific antibody at a dose of 0.03 mg / kg showed a significant inhibitory effect on tumor volume, with statistical significance (P<0.001) . At doses of 0.03 and 0.01 mg / kg, the tumor growth inhibition rates (TGITV) of the bispecific antibody 5-7G were 81.59%and 21.99%, respectively, indicating that the bispecific antibody with the 5-7G structure can effectively inhibit tumor growth at a lower dose of 0.03 mg / kg.
[0653] Table 21: Effect of CD3 / CAIX Bispecific Antibody (5-7G Structure Molecule) on Tumor Volume in OSRC2 Immune-Reconstituted Model NCG Mice
[0654] Example 4: Preparation of Masked CD3 / CAIX / CAIX Bispecific Antibodies
[0655] Based on the murine CD3 antibody SP-34, the humanized CD3 antibody TA1 was obtained through mutation, library construction, humanization, and multiple screenings. The CAIX single-domain antibody is referred to as anti-CAIX VHH in the structural diagram in FIG. 3.
[0656] According to FIG. 3, the structure of the bispecific antibody CAIX-BsAb based on TA1-Fab (monovalent for CD3) and CAIX VHH (bivalent for CAIX) was designed. In the TA1-Fab, TA1-Fab-VH is located at the N-terminus of CH1, and TA1-Fab-VL is located at the N-terminus of CL. In FIG. 3, “Anti-CD3 Fab” represents any one of the anti-CD3 Fabs described herein, including the anti-CD3 Fabs (from 153-Ab1 to 153-Ab36) in Tables 10-11; “CA IX VHH” represents any one of the anti-CAIX VHHs described herein, including the anti-CAIX VHHs (from 193-Ab1 to 193-Ab20) in Table 14.; “anti-CD3 mask” represents any one of the anti-CD3 masks described herein, including any one of the anti-CD3 masks (from ma-153-1 to ma-153-17) in Table 12. The single-domain antibody CAIX VHH is located at the C-terminus of TA1-Fab. Using a cleavable substrate linker as a connector, an anti-CD3 mask is incorporated into the bispecific antibody by linking it to the N-terminus of TA1-Fab. For example, as shown in FIG. 3-3, in one embodiment, the heavy chain of the masked CD3 / CAIX / CAIX bispecific antibody is: CD3 mask + linker + CD3-Fab-VH-CH1 + anti-CAIX VHH + anti-CAIX VHH; the light chain is: CD3-Fab-VL-CL. In this embodiment, TA1F represents the Fab region of the anti-CD3 antibody clone TA1, where TA1 VH is connected to CH1 (IgG4) , and TA1 VL is connected to CL (kappa) . The light chain is a common kappa light chain, and CH1 is derived from IgG4.
[0657] In some embodiments, the structure of the bispecific antibodies adopt the above-mentioned design based on the CD3-Fab framework (FIG. 3-3) . Plasmids were constructed and co-transfected into 293F cells via PEI-mediated transfection. After protein expression and purification, CD3 / CAIX bispecific antibodies with a purity of ≥90%were obtained.
[0658] Example 5: The masked CD3 / CAIX / CAIX bispecific antibodies induce T cells to kill CAIX-positive tumor target cells
[0659] To evaluate the functional activity of the bispecific antibodies, a tumor cell line killing assay was performed. The killing effect of T cells induced by the bispecific antibodies on target cells was assessed in human colorectal cancer cells HT-29 (with moderate CAIX expression) . Human PBMCs were used as effector cells, and the killing was detected after incubating with the antibodies for 72 hours.
[0660] The tumor cells were digested with trypsin / EDTA, washed once with pre-cooled PBS, and resuspended in RPMI 1640 medium containing 10%FBS. The cells were then seeded at a density of 5,000 cells / well in flat-bottom 96-well plates (Corning 3599) . After 4 hours, 50 μL of serially diluted antibody solutions (including serially diluted naked CD3 / CAIX / CAIX antibody, masked CD3 / CAIX / CAIX bispecific antibody, and masked CD3 / CAIX / CAIX bispecific antibody treated with matriptase (MTSP1) or urokinase (uPa) protease) were added, with three replicates for each condition. The plates were incubated for over 30 minutes to allow the proteins to fully bind to the cells. PBMCs were then resuscitated and added to 10 mL of RPMI 1640 medium containing 10%FBS. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were thoroughly resuspended. The cell density was adjusted according to the experimental requirements, and PBMCs were added to the target cells at 50 μL per well to reach a final E: T ratio of 0.5: 1. The 96-well plates were incubated at 37℃ in a 5%CO2 incubator for 3 days. Finally, cell viability was detected using the CCK8 assay (Dojindo, CK04-20) , and the cell survival rate was calculated using GraphPad Prism software. The experimental results are expressed as mean ± standard deviation (Mean ± SD) or killing EC50 (nM) .
[0661] The masked CD3 / CAIX / CAIX bispecific antibody exhibited a reduced killing effect on tumor cells compared to the naked CD3 / CAIX / CAIX bispecific antibody, due to its masking effect. When the mask is removed using proteases, the killing effect was restored to levels similar to the naked antibody. The experimental results are shown in the table below. This indicates that the masking mechanism effectively reduced the antibody's killing activity until it is unmasked by proteases, potentially enhancing its safety profile by limiting off-target effects. 5-7G and 3-3 have the same anti-CD3 Fab and anti-CAIX VHH sequences. Different mask sequences (e.g., Table 12) and linker sequences (e.g., Table 13) were tested and the results showed all mask sequences can provide masking effects and all linker sequences allow effective cleavage and restoration of the killing effects.
[0662] Table 22: Killing Effect of Drugs on HT-29 Tumor Cells (EC50, nM)
[0663] OTHER EMBODIMENTS
[0664] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
1.An antigen-binding protein, comprising:(a) a first antigen-binding domain (B1) comprising a Fab and a first mask peptide (M1) , wherein the Fab comprises a first chain and a second chain, wherein the first mask peptide (M1) impairs the binding of the first antigen-binding domain (B1) to the target of the first antigen-binding domain (B1) , and the first mask peptide (M1) is linked to the first antigen-binding domain (B1) via a first cleavable linker (L1) ;(b) a second antigen-binding domain (B2) , wherein the second antigen-binding domain (B2) comprises a first VHH that is linked to C-terminus of the first or second chain of the Fab; and(c) a half-life extension unit (E) , wherein the half-life extension unit (E) is linked to N-terminus of the first mask peptide (M1) .2.The antigen-binding protein of claim 1, wherein:(1) the first chain of the Fab comprises a heavy chain variable region (VH) and a CH1 domain and the second chain of the Fab comprises a light chain variable region (VL) and a CL domain;(2) the first chain of the Fab comprises a VL and a CL domain and the second chain of the Fab comprises a VH and a CH1 domain;(3) the first chain of the Fab comprises a VH and a CL domain and the second chain of the Fab comprises a VL and a CH1 domain; or(4) the first chain of the Fab comprises a VL and a CH1 domain and the second chain of the Fab comprises a VH and a CL domain.3.The antigen-binding protein of claim 2, wherein the first cleavable linker (L1) is linked to N-terminus of the VH or the VL.4.The antigen-binding protein of any one of claims 1-3, wherein the antigen-binding protein further comprises a second VHH, optionally the first and second VHHs are the same.5.The antigen-binding protein of claim 4, wherein:(1) the first VHH is linked to C-terminus of the first chain of the Fab and the second VHH is linked to C-terminus of second chain of the Fab;(2) the first VHH is linked to C-terminus of the second chain of the Fab and the second VHH is linked to C-terminus of first chain of the Fab;(3) the first VHH is linked to the C-terminus of the first chain of the Fab and the second VHH is linked to the C-terminus of the first VHH; or(4) the first VHH is linked to the C-terminus of the second chain of the Fab and the second VHH is linked to the C-terminus of the first VHH.6.The antigen-binding protein of any one of claims 1-5, wherein the first antigen-binding domain (B1) specifically binds to an immune cell receptor antigen (e.g., CD3, 4-1BB (CD137) , 4-1BB-L, CD28, CD40, CD40-L, CD58, CD2, or CD8) and the second antigen-binding domain (B2) specifically binds to a tumor antigen (e.g., CAIX) .7.The antigen-binding protein of any one of claims 1-6, wherein first antigen-binding domain (B1) specifically binds to CD3.8.The antigen-binding protein of any one of claims 1-7, wherein first antigen-binding domain (B1) specifically binds to a CD3 positive T cell.9.The antigen-binding protein of any one of claims 1-8, wherein the second antigen-binding domain (B2) specifically binds to CAIX.10.The antigen-binding protein of any one of claims 1-9, wherein the second antigen-binding domain (B2) specifically binds to a CAIX positive tumor cell.11.The antigen-binding protein of any one of claims 1-10, wherein the first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.12.The antigen-binding protein of claim 11, wherein the first mask peptide (M1) is linked to the half-life extension unit (E) via a linker that comprises the amino acid sequence of any one of SEQ ID NOs: 273-288.13.The antigen-binding protein of any one of claims 1-12, wherein the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.14.The antigen-binding protein of any one of claims 1-13, wherein the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .15.The antigen-binding protein of claim 14, wherein the VHH that specifically binds to human serum albumin (HSA) comprises the amino acid sequence of SEQ ID NO: 268.16.An antigen-binding protein comprising: (1) a CD3-binding domain, and (2) a CAIX-binding domain, wherein the CD3-binding domain comprises a Fab comprising a heavy chain variable region (VH) and a light chain variable region (VL) , and the CAIX-binding domain comprises a VHH, wherein the CAIX-binding domain is linked to C-terminus of the CD3-binding domain.17.The antigen-binding protein of claim 16, wherein the CD3-binding domain specifically binds to a CD3 positive T cell.18.The antigen-binding protein of claim 16 or 17, wherein the CAIX-binding domain specifically binds to a CAIX positive tumor cell .19.The antigen-binding protein of any one of claims 16-18, wherein the CAIX-binding domain comprises two VHHs linked in tandem.20.The antigen-binding protein of any one of claims 16-19, wherein the CAIX-binding domain comprises a VHH that comprises:(a) the amino acid sequence of any one of SEQ ID NOs: 238-257;(b) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the amino acid sequence of any one of SEQ ID NOs: 238-257; or(c) an amino acid sequence that has one or more additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 238-257, wherein the additions, deletions and / or substitutions do not occur in a CDR region.21.The antigen-binding protein of claim 16, wherein the antigen-binding protein further comprises a first mask peptide (M1) that impairs the binding of the CD3-binding domain to the target of the CD3-binding domain (e.g., CD3) , and the first mask peptide is linked to the CD3-binding domain via a first cleavable linker (L1) .22.The antigen-binding protein of claim 21, wherein first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.23.The antigen-binding protein of claim 21 or 22, wherein the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.24.The antigen-binding protein of any one of claims 21-23, wherein the antigen-binding protein further comprises a half-life extending unit (E) .25.The antigen-binding protein of claim 24, wherein the half-life extending unit (E) is linked to the N-terminus of the first mask peptide (M1) via a linker that comprises the amino acid sequence of any one of SEQ ID NOs: 273-288.26.The antigen-binding protein of claim 24 or 25, wherein the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .27.The antigen-binding protein of claim 26, wherein the VHH that specifically binds to human serum albumin (HSA) comprises the amino acid sequence of SEQ ID NO: 268.28.An antigen-binding protein, comprising:(a) a first peptide comprising, from the N-terminus to the C-terminus, a half-life extension unit (E) , a first mask peptide (M1) , a first cleavable linker (L1) , a VH, a CH1, a first VHH that specifically binds to a first antigen; and(b) a second peptide comprising, from the N-terminus to the C-terminus, a VL, and a CL,wherein the VH and VL form a Fab that specifically binds to a second antigen, and the first mask peptide (M1) impairs the binding of the Fab to the second antigen.29.The antigen binding protein of claim 28, wherein the first peptide further comprises a second VHH that is linked to the C-terminus of the first VHH.30.The antigen-binding protein of claim 29, wherein the first and second VHHs are the same.31.The antigen binding protein any one of claims 28-30, wherein the first antigen is CAIX and the second antigen is CD3.32.The antigen binding protein any one of claims 28-31, wherein the VH comprises any one of the combinations of the HCDR1, HCDR2, and HCDR3 sequences in Tables 1, 3, and 5, and the VL comprises any one of the combinations of the LCDR1, LCDR2, and LCDR3 sequences in Tables 2, 4, and 6.33.The antigen binding protein any one of claims 28-32, wherein the VH comprises any one of the amino acid sequences in Table 7, and the VL comprise any one of the amino sequences in Table 8.34.The antigen binding protein any one of claims 28-33, wherein the first VHH comprises any one of the combinations of the CDR1, CDR2, and CDR3 sequences in Tables 15-17.35.The antigen binding protein any one of claims 28-34, wherein the half-life extending unit (E) comprises a VHH that specifically binds to human serum albumin (HSA) .36.The antigen binding protein any one of claims 28-35, wherein the first mask peptide (M1) comprises the amino acid sequence of any one of SEQ ID NOs: 150-166.37.The antigen binding protein any one of claims 28-36, wherein the first cleavable linker (L1) comprises a first cleavable peptide (C1) , wherein the first cleavable peptide (C1) comprises the amino acid sequence of any one of SEQ ID NOs: 258-267.38.A nucleic acid comprising a polynucleotide encoding the antigen-binding protein of any one of claims 1-37.39.The nucleic acid of claim 38, wherein the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA) .40.A vector comprising one or more of the nucleic acids of claim 38 or 39.41.A cell comprising the vector of claim 40.42.The cell of claim 41, wherein the cell is a HEK293 cell or CHO cell.43.A cell comprising one or more of the nucleic acids of claim 38 or 39.44.A method of producing an antigen-binding protein, the method comprising(a) culturing the cell of any one of claims 41-43 under conditions sufficient for the cell to produce the antigen-binding protein; and(b) collecting the antigen-binding protein produced by the cell.45.A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antigen-binding protein of any one of claims 1-37, to the subject.46.A method of decreasing the rate of tumor growth, the method comprisingcontacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein of any one of claims 1-37.47.A method of killing a tumor cell, the method comprisingcontacting a tumor cell with an effective amount of a composition comprising the antigen-binding protein of any one of claims 1-37.48.A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-37, and a pharmaceutically acceptable carrier.49.A chimeric antigen receptor (CAR) comprising the antigen-binding protein of any one of claims 1-37.50.An antibody-drug conjugate (ADC) comprising the antigen-binding protein of any one of claims 1-37, covalently bound to a therapeutic agent.51.The antibody-drug conjugate of claim 41, wherein the therapeutic agent is a cytotoxic or cytostatic agent.52.A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-37, the CAR of claim 39, or the antibody-drug conjugate of claim 50 or 51, and a pharmaceutically acceptable carrier.53.A method of treating a subject having a disease, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition of claim 52 to the subject.54.The method of claim 53, wherein the disease is cancer; preferably, the cancer is selected from the group consisting of KIRC (Kidney Renal Clear Cell Carcinoma) , CHOL (Cholangio carcinoma) , PAAD (Pancreatic adenocarcinoma) , LUSC (Lung squamous cell carcinoma) , LUAD (Lung adenocarcinoma) , HNSC (Head and Neck squamous cell carcinoma) , BLCA (Bladder Urothelial Carcinoma) , ESCA (Esophageal carcinoma) , COAD (Colon adenocarcinoma) , READ (Rectum adenocarcinoma) , SCLC (Small cell lung cancer) , STAD (Stomach adenocarcinoma) , MESO (Mesothelioma) and CESC (Cervical squamous cell carcinoma) .55.The method of claim 53, wherein the subject has a CAIX-positive cancer, e.g., cervical cancer, renal cancer, brain cancer, head and neck cancer, esophageal cancer, intestinal cancer, breast cancer, ovarian cancer, endometrial cancer, or bladder cancer.
Citation Information
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