Novel bispecific polypeptide complex

Abstract

The present disclosure relates to the field of antibody drugs, and specifically relates to novel antibodies replacing CH1 and CL domains with specific protein pairs, and uses of the novel antibodies.

Description

Novel bispecific polypeptide complexes

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411305665.3, filed on September 18, 2024, and Chinese Patent Application No. 202511310926.5, filed on September 15, 2025, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of antibody drugs. Specifically, it includes novel antibodies using specific proteins to replace CH1 and CL domains and their applications. BACKGROUND

[0004] With the development of antibody technology, more and more monoclonal antibodies have been approved for the treatment of diseases including malignant tumors, autoimmune diseases, viral infections, etc. Monoclonal antibody drugs act through a single mechanism, such as inhibiting the growth of tumors through a single signaling pathway. Since there are many mechanisms of immune escape, monoclonal antibody drugs are prone to drug resistance in clinical practice, thereby weakening the therapeutic effect of monoclonal antibodies. In this context, bispecific antibodies have emerged and gradually become a new research direction. Through genetic engineering, antibody fragments targeting two targets are combined together, so that one antibody can bind to two different antigens or two different epitopes of the same antigen, thereby achieving better therapeutic effect.

[0005] In order to achieve the purpose of simultaneously targeting two or more antigens, natural bispecific antibodies need to be composed of multiple polypeptide complexes, such as two heavy chains plus two light chains to assemble into an IgG-like antibody general form. Such molecules cannot be correctly assembled like normal IgG, and in the absence of modification, by-products containing homodimers and light chain mismatches or deletions, etc. Due to the similarity of their physicochemical characteristics, these by-products are often difficult to separate from the target product through conventional purification steps or require compromises in yield. This undoubtedly significantly increases the difficulty and cost of producing bispecific antibodies.

[0006] At present, many forms of recombinant bispecific antibodies have been developed, and the 2+2 form of bispecific antibodies obtained by fusing and expressing single-chain domains such as ScFv / VHH domains on heavy chains or light chains (Coloma, MJ et al., Nature Biotech 15 (1997) 159-163; Morrison, S.L. Nature Biotech. 25 (2007) 1233-1234). The ScFv / VHH single-chain domains introduced in symmetrical bispecific antibodies often bring additional challenges to the drugability and stability of antibodies. Therefore, there are also various technologies to try to solve the mispairing problem in asymmetric IgG-like antibody structures.

[0007] By introducing mutations into the Fc region, such as knobs-into-holes (Ridgway et al., Protein Engineering, 9(7), pp. 617-21 (1996); Merchant et al., Nature Biotechnology, 16(7), pp. 677-681 (1998)), electrostatics (Gunasekaran et al., Journal of Biological Chemistry, 285(25), pp. 19637-19646 (2010)), or negative design (Kreudenstein et al., mAbs, 5(5), pp. 646-654 (2013); Leaver-Fay et al., Structure, 24(4), pp. 641-651 (2016)), the mispairing between different heavy chains is controlled or made easy to remove.

[0008] However, there are still challenges in selective pairing between light and heavy chains. Current engineering approaches for light chain mispairing mainly focus on the constant domains between light and heavy chains (CH1-CL). These include the CrossMab platform that swaps the CH1 and CL domains (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp. 11187-11192 (2011)), the introduction of disulfide bonds at the orthogonal interface (Mazor et al., mAbs, 7(2), pp. 377-389 (2015)), charge modification in the CH1-CL region (Liu et al., Journal of Biological Chemistry, 290(12), pp. 7535-7562 (2015)), or the introduction of various mutations in the variable and constant regions (Lewis et al., Nature Biotechnology, 32(2), pp. 191-198 (2014)) and (Dillon et al., mAbs, 9(2), pp. 213-230 (2017)), and the use of TCR constant regions to replace the CH1-CL region (WO2019057122A1), etc. These strategies require optimization of the light-heavy chain ratio to maximize the correct pairing between light and heavy chains.

[0009] Therefore, it is necessary to design a bispecific / multispecific format with ideal light-heavy chain pairing selectivity to improve its expression, stability and purity. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figures 1, 2a, 2b, 3a and 3b show the affinity of CH / CL1 replaced monoclonal and bispecific antibodies to cells detected by flow cytometry, wherein the flow cytometry analysis results of the samples on NCI-H1975 cells are shown respectively;

[0011] Figures 4, 5, 6a and 6b show the analysis graphs of CH / CL1 replaced monoclonal and bispecific antibody internalization experiment data, wherein the antibody internalization assay results of the samples on NCI-H1975 cells are shown respectively. SUMMARY

[0012] I. Polypeptide Complex

[0013] For the above purpose, the present disclosure provides a polypeptide complex consisting of two pairs of polypeptides, comprising a first pair of polypeptides and a second pair of polypeptides, each pair of polypeptides comprising or consisting of a heavy chain and a light chain, wherein the heavy chain comprises a VH and a CH1 domain, and the light chain comprises a VL and a CL domain, the VH and VL domains of the first pair of polypeptides can form a first Fv fragment that specifically binds to a first antigen, and the VH and VL domains of the second pair of polypeptides can form a second Fv fragment that specifically binds to a second antigen, wherein at least one pair of interaction domains is introduced into the first pair of polypeptides and / or the second pair of polypeptides, and each pair of interaction domains can form a dimer by itself or in combination with other proteins, wherein the interaction domain is a homodimerization / heterodimerization domain.

[0014] In some embodiments, the introduction of the interaction domain does not affect the properties of the polypeptide complex, such as monospecific antibody or bispecific antibody or multispecific antibody, before the introduction, such as its affinity or endocytosis activity.

[0015] In some embodiments, the at least one pair of interaction domains is selected from a pair of PD1 chain and PDL1 chain, a pair of PD1 chain and PDL2 chain, and a pair of CD47 chain and SIRPa chain.

[0016] In some embodiments, the at least one pair of interaction domains is selected from a pair of PD1 chain and PDL1 chain, a pair of PD1 chain and PDL2 chain, and a pair of CD47 chain and SIRPa chain.

[0017] In some embodiments, the present disclosure provides a polypeptide complex comprising a first antigen binding moiety, the first antigen binding moiety comprising a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH1), the VH1 operably linked to a first domain (Domain 1); and a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable region (VL1), the VL1 operably linked to a second domain (Domain 2),

[0018] wherein the first antigen binding moiety specifically binds to a first antigen, wherein the first domain and the second domain are capable of interacting to form a dimer.

[0019] In some embodiments, the first domain is used to replace the antibody heavy chain constant region CH1, and the second domain is used to replace the antibody light chain constant region CL.

[0020] In some embodiments, in the polypeptide complex of the present disclosure, the VH1 and VL1 of the first antigen binding moiety form a first antigen binding site that specifically binds to the first antigen.

[0021] In some embodiments, the polypeptide complex is monospecific, comprising two first antigen binding moieties, and optionally the first antigen binding moieties are each operably linked to a dimerization domain, e.g., an Fc region.

[0022] In some embodiments, the polypeptide complex is multispecific, e.g., bispecific, comprising at least a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first polypeptide and a second polypeptide as described above.

[0023] In some embodiments, the polypeptide complex is bispecific, comprising a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first polypeptide and a second polypeptide as described above.

[0024] In some embodiments, the second antigen binding moiety comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2).

[0025] In some embodiments, the VH2 and VL2 of the second antigen binding moiety form a second antigen binding site that specifically binds to a second antigen.

[0026] In some embodiments, the second antigen binding moiety comprises:

[0027] a third polypeptide comprising, from N-terminus to C-terminus, VH2 operably linked to a third domain (Domain 3) comprising CH1 or being CH1; and a fourth polypeptide comprising, from N-terminus to C-terminus, VL2 operably linked to a fourth domain (Domain 4) comprising CL or being CL.

[0028] In some embodiments, the CH1 region is a human IgG CH1, e.g., a human IgGl CH1, a human IgG2 CH1, a human IgG3 CH1, or a human IgG4 CH1. In one embodiment, the CH1 region comprises or consists of the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence at least 90% identical, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0029] In some embodiments, the CL region is a human Kappa light chain CL region or a human Lamda light chain CL region. In some embodiments, the CL region comprises or consists of the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence at least 90% identical, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0030] In some embodiments, in the polypeptide complex of the present disclosure, the VH1 of the first antigen binding moiety is not readily mispaired with the VL2 of the second antigen binding moiety, and / or the VH2 of the second antigen binding moiety is not readily mispaired with the VL1 of the first antigen binding moiety.

[0031] In some embodiments, in the polypeptide complex of the present disclosure, the first antigen binding moiety is further operably linked to a first dimerization domain, the second antigen binding moiety is further operably linked to a second dimerization domain, and the first and second dimerization domains associate. In some embodiments, the first and second dimerization domains are Fc regions, respectively.

[0032] The present disclosure describes different structures or domains or features that make up the polypeptide complex, and it should be understood that the various structures or domains or features described can be combined in any combination to make the polypeptide complex of the present disclosure.

[0033] I. First and second domains

[0034] In some embodiments, in the polypeptide complex of the present disclosure, the first and second domains form a dimer through a native interchain bond and / or a non-native interchain bond.

[0035] In some embodiments, the first and second domains form a dimer through a native interchain bond.

[0036] In some embodiments, in the polypeptide complex of the present disclosure, the first and second domains form a dimer through at least one non-native interchain bond.

[0037] In some embodiments, the non-native interchain bond is formed between a particular mutant residue in the first domain and a particular mutant residue in the second domain.

[0038] In some embodiments, at least one of the particular mutant residue in the first domain and the particular mutant residue in the second domain is a cysteine residue, or both the particular mutant residues in the first and second domains that form a disulfide bond are cysteine residues.

[0039] In some embodiments, the non-native interchain bond is a disulfide bond. In some embodiments, wherein the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-native interchain bonds.

[0040] In some embodiments, in the polypeptide complex of the present disclosure, the particular mutant residue in the first domain and the particular mutant residue in the second domain are located at a contact interface of the first and second domains.

[0041] In some embodiments, the first domain or the second domain comprises a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus.

[0042] In some embodiments, the N-terminal linker is a N-terminal fragment of the molecule from which the first domain or the second domain is derived, and / or the C-terminal linker is a C-terminal fragment of the molecule from which the first domain or the second domain is derived.

[0043] In some embodiments, the N-terminal linker comprised by the first domain is derived from a N-terminal fragment of the heavy chain constant region CHI, and / or the C-terminal linker comprised by the first domain is derived from a C-terminal fragment of the heavy chain constant region CHI.

[0044] In some embodiments, the N-terminal linker comprised by the second domain is derived from a N-terminal fragment of the light chain constant region CL, and / or the C-terminal linker comprised by the second domain is derived from a C-terminal fragment of the light chain constant region CL.

[0045] In some embodiments, the first domain comprises a PD1 chain and the second domain comprises a domain capable of interacting with a PD1 chain, such as a PDL1 chain or a PDL2 chain.

[0046] In some embodiments, the first domain comprises a domain capable of interacting with a PD1 chain, such as a PDL1 chain or a PDL2 chain, and the second domain comprises a PD1 chain.

[0047] In some embodiments, the first domain comprises a PDL1 or PDL2 chain and the second domain comprises a domain capable of interacting with a PDL1 or PDL2 chain, such as a PD1 chain.

[0048] In some embodiments, the first domain comprises a domain capable of interacting with a PDL1 or PDL2 chain, such as a PD1 chain, and the second domain comprises a PDL1 or PDL2 chain.

[0049] In some embodiments, the first domain comprises a PD1 chain and the second domain comprises a PDL1 or PDL2 chain; or the first domain comprises a PDL1 or PDL2 chain and the second domain comprises a PD1 chain.

[0050] In some embodiments, the first domain comprises a CD47 chain and the second domain comprises a domain capable of interacting with a CD47 chain, such as a SIRPa chain.

[0051] In some embodiments, the first domain comprises a domain capable of interacting with a CD47 chain, such as a SIRPa chain, and the second domain comprises a CD47 chain.

[0052] In some embodiments, the first domain comprises a SIRPa chain and the second domain comprises a domain capable of interacting with a SIRPa chain, e.g., a CD47 chain.

[0053] In some embodiments, the first domain comprises a domain capable of interacting with a SIRPa chain, e.g., a CD47 chain, and the second domain comprises a SIRPa chain.

[0054] In some embodiments, the first domain comprises a CD47 chain and the second domain comprises a SIRPa chain; or the first domain comprises a SIRPa chain and the second domain comprises a CD47 chain.

[0055] Different protein chains suitable for use as the first domain or the second domain of the polypeptide complex of the present disclosure are described below.

[0056] PD1 chain

[0057] In some embodiments, the PD1 chain is from the extracellular immunoglobulin variable region (IgV)-like domain of a PD1 protein, which can bind to PDL1 and / or PDL2. In some embodiments, the PD1 chain is a wild-type IgV-like domain or a fragment of a wild-type IgV-like domain, e.g., comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0058] In some embodiments, the PD1 chain used in the polypeptide complex of the present disclosure is a modified PD1 chain having mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parent PD1 chain.

[0059] In some embodiments, the parent PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. When referring to the mutation positions of the modified PD1 chain herein, the mutation positions are all sequentially numbered positions relative to the sequence of SEQ ID NO: 1, i.e., taking the first amino acid corresponding to the first position of SEQ ID NO: 1 as the first position start, regardless of whether it also comprises a linker at the N-terminus and / or C-terminus.

[0060] In some embodiments, the modified PD1 chain suitable for use in the polypeptide complex of the present disclosure has a cysteine mutation (i.e., mutated to cysteine) to generate an interchain disulfide bond, or a mutation of a native cysteine to a non-cysteine, e.g., S, and / or an N-glycosylation site mutation (i.e., mutation of an N-glycosylation site to a non-glycosylated amino acid, e.g., S or D), preferably the N-glycosylation site mutation does not affect its interchain disulfide bond formation and / or binding of the PD1 chain to the PDL1 chain or PDL2 chain, as compared to the wild-type IgV-like domain fragment, e.g., SEQ ID NO: 1.

[0061] In some embodiments, the modified PD1 chain suitable for use in the polypeptide complex of the present disclosure has one or more amino acid residue mutations selected from position 15, 24, 30, 40, 44, 59, or 82, wherein the modified PD1 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 1, as compared to the wild-type IgV-like domain fragment, e.g., SEQ ID NO: 1.

[0062] In some embodiments, the modified PD1 chain comprises a glycosylation site mutation, e.g., mutation of N at position 15, 24, 40, or 82 to a non-glycosylated amino acid, e.g., S or D.

[0063] In some embodiments, the modified PD1 chain comprises a mutation to cysteine at position 30 and / or 44, or a mutation of cysteine at position 59 to a non-cysteine, e.g., S.

[0064] In some embodiments, the modified PD1 chain has amino acid residue mutations at positions 15, 24, 40, 59, and 82, and optionally one or more amino acid residue mutations (e.g., amino acid residue mutations at position 30 or 44), wherein the modified PD1 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 1.

[0065] In some embodiments, the modified PD1 chain has

[0066] amino acid residue mutations at positions 15, 24, 40, 44, 59, and 82,

[0067] amino acid residue mutations at positions 15, 24, 30, 40, 59, and 82, or

[0068] amino acid residue mutations at positions 15, 24, 40, 59, and 82, wherein the PD1 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 1.

[0069] In some embodiments, the amino acid residue at position 15, 24, 40, or 59 is mutated to S, the amino acid residue at position 30 is mutated to C, the amino acid at position 44 is mutated to T or C, and / or the amino acid at position 82 is mutated to D.

[0070] In some embodiments, the modified PD1 chain has one or more amino acid residue mutations selected from 15S, 24S, 30C, 40S, 44C / T, 59S, or 82D, wherein the modified PD1 chain mutation site is an ordinal number position relative to the sequence of SEQ ID NO: 1.

[0071] In some embodiments, the modified PD1 chain has a combination of mutations of N15S, N24S, N40S, C59S, N82D, and optionally one or more other mutations, such as 30C, and / or 44T / C.

[0072] In some embodiments, the modified PD1 chain has a combination of mutations of:

[0073] N15S, N24S, N40S, K44C, C59S, N82D;

[0074] N15S, N24S, V30C, N40S, C59S, N82D;

[0075] N15S, N24S, N40S, K44T, C59S, N82D; or

[0076] N15S, N24S, N40S, C59S, N82D.

[0077] In some embodiments, the modified PD1 chain suitable for use in the polypeptide complex of the present disclosure comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 8-11, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 8-11.

[0078] In some embodiments, the modified PD1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 8, and comprises N15S, N24S, N40S, K44C, C59S, N82D. In some embodiments, the modified PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8.

[0079] In some embodiments, the modified PD1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 9, and comprises N15S, N24S, V30C, N40S, C59S, N82D. In some embodiments, the modified PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9.

[0080] In some embodiments, the modified PD1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10, and comprises N15S, N24S, N40S, K44T, C59S, N82D. In some embodiments, the modified PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10.

[0081] In some embodiments, the modified PD1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 11, and comprises N15S, N24S, N40S, C59S, N82D. In some embodiments, the modified PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0082] In some embodiments, the PD1 chain suitable for use in the polypeptide complexes of the present disclosure is a PD1 chain comprising a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus, e.g., a modified PD1 chain comprising a N-terminal and / or C-terminal linker.

[0083] In some embodiments, the PD1 chain suitable for use in the polypeptide complexes of the present disclosure comprises a N-terminal linker at its N-terminus and is linked to a heavy chain variable region or a light chain variable region via the N-terminal linker. In some embodiments, the N-terminal linker is selected from the group consisting of: the N-terminal contiguous segment of the PD1 chain in the protein from which the PD1 chain is derived; the N-terminal segment of a heavy chain constant region CH1 (e.g., when it is linked to a heavy chain variable region via the N-terminus), the N-terminal segment of a light chain constant region CL (e.g., when it is linked to a light chain variable region via the N-terminus), or (G4S) nwherein n = 1-10, e.g., 1, 2, or 3. In some embodiments, the N-terminal flanking segment of the PD1 chain is DRPWNP (SEQ ID NO: 12). In some embodiments, the N-terminal segment of the heavy chain constant region is ASTKGP (SEQ ID NO: 31). In some embodiments, the N-terminal segment of the light chain constant region is RTVAAP (SEQ ID NO: 30). In some embodiments, the N-terminal linker is selected from the group consisting of DRPWNP, RTVAAP, ASTKGP, (G4S)i, and (G4S)2.

[0084] In some embodiments, the PD1 chain comprises a C-terminal linker at the C-terminus. In some embodiments, the C-terminal linker is selected from the group consisting of the C-terminal flanking segment of the PD1 chain in the protein from which the PD1 chain is derived; the C-terminal segment of the heavy chain constant region CHI (e.g., when it is N-terminally connected to a heavy chain variable region), the C-terminal segment of the light chain constant region (e.g., when it is N-terminally connected to a light chain variable region), or (G4S)n, wherein n = 1-10, e.g., 1, 2, or 3. In some embodiments, the C-terminal segment of the PD1 chain is selected from the group consisting of ERRAE (SEQ ID NO: 13), ERRAESC (SEQ ID NO: 21), or ERRGEC (SEQ ID NO: 22). In some embodiments, the C-terminal linker is selected from the group consisting of ERRAE, ERRAESC, ERRGEC, GE, (G4S)i, and (G4S)2.

[0085] In some embodiments, the PD1 chain comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at its N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13), ERRAESC (SEQ ID NO: 21), or ERRGEC (SEQ ID NO: 22) at its C-terminus.

[0086] In some embodiments, the modified PD1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2-7, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2-7. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, 21, or 22.

[0087] In some embodiments, the modified PD1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, and comprises N15S, N24S, N40S, K44C, C59S, N82D. In some embodiments, the PD1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0088] In some embodiments, the modified PD1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 3, and comprises N15S, N24S, V30C, N40S, C59S, N82D. In some embodiments, the PD1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0089] In some embodiments, the modified PD1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4 or 5, and comprises N15S, N24S, N40S, K44T, C59S, N82D. In some embodiments, the PD1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0090] In some embodiments, the modified PD1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7, and comprises N15S, N24S, N40S, C59S, N82D. In some embodiments, the PD1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6 or 7.

[0091] PDL1 chain

[0092] In some embodiments, the PDL1 chain is from an extracellular immunoglobulin variable region (IgV)-like domain of a PDL1 protein, which can bind to PD1. In some embodiments, the PDL1 chain is a wild-type IgV-like domain or a fragment of a wild-type IgV-like domain, for example, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0093] In some embodiments, the PDL1 chain used in the polypeptide complex of the present disclosure is a modified PDL1 chain having mutations compared to a fragment of a wild-type IgV-like domain, for example, a parent PDL1 chain.

[0094] In some embodiments, the parent PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. When referring to the mutation positions of a modified PDL1 chain herein, the mutation positions are all sequentially numbered positions relative to the sequence of SEQ ID NO: 14, i.e., taking the first amino acid corresponding to the first position of SEQ ID NO: 14 as the first position, regardless of whether it also comprises a linker at the N-terminus and / or C-terminus.

[0095] In some embodiments, the modified PDL1 chain suitable for use in the polypeptide complex of the present disclosure has a cysteine mutation (i.e., mutated to cysteine) to generate an interchain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as T), preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or the binding of the PDL1 chain to the PD1 chain, compared to a fragment of a wild-type IgV-like domain, for example, SEQ ID NO: 14.

[0096] In some embodiments, the modified PDL1 chain has one or more amino acid residue mutations selected from positions 4, 17, 23, 73, or 104, wherein the mutation positions of the modified PDL1 chain are sequentially numbered positions relative to the sequence of SEQ ID NO: 14.

[0097] In one embodiment, the modified PDL1 chain has amino acid mutations at positions 17 and 104, wherein the mutation positions of the modified PDL1 chain are sequentially numbered positions relative to the sequence of SEQ ID NO: 14.

[0098] In one embodiment, the modified PDL1 chain has amino acid mutations at positions 4, 17, 23, and 73, wherein the mutation positions of the modified PDL1 chain are sequentially numbered positions relative to the sequence of SEQ ID NO: 14.

[0099] In one embodiment, the fourth amino acid residue is mutated to A, the seventeenth amino acid residue is mutated to T, the twenty-third amino acid residue is mutated to A, the seventy-third amino acid residue is mutated to A, and / or the one hundred and fourth amino acid residue is mutated to C.

[0100] In some embodiments, the modified PDL1 chain has one or more amino acid residue mutations selected from 4A, 17T, 23A, 73A, and 104C, wherein the PDL1 chain mutation site is the sequentially numbered site relative to the sequence of SEQ ID NO: 14.

[0101] In some embodiments, the modified PDL1 chain has an amino acid residue mutation of 17T, and optionally one or more other amino acid mutations, e.g., 104C, 4A, 23A, or 73A, wherein the PDL1 chain mutation site is the sequentially numbered site relative to the sequence of SEQ ID NO: 14.

[0102] In some embodiments, the modified PDL1 chain has an amino acid residue mutation selected from 4A, 17T, 23A, and 73A, wherein the PDL1 chain mutation site is the sequentially numbered site relative to the sequence of SEQ ID NO: 14.

[0103] In some embodiments, the modified PDL1 chain has a combination of mutations of N17T, D104C; or N17T, T4A, K23A, Q73A.

[0104] In some embodiments, the modified PDL1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 19 or 20, or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 20.

[0105] In some embodiments, the modified PDL1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, and comprises N17T, D104C. In some embodiments, the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19.

[0106] In some embodiments, the modified PDL1 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and comprises N17T, T4A, K23A, Q73A. In some embodiments, the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0107] In some embodiments, the PDL1 chain suitable for use in a polypeptide complex of the disclosure is a PDL1 chain comprising a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus, e.g., a modified PDL1 chain comprising a N-terminal and / or C-terminal linker.

[0108] In some embodiments, the PDL1 chain suitable for use in a polypeptide complex of the disclosure comprises a N-terminal linker at its N-terminus and is linked to a heavy chain variable region or a light chain variable region via the N-terminal linker. In some embodiments, the N-terminal linker is selected from the group consisting of: the N-terminal contiguous segment of the PDL1 chain in the protein from which the PDL1 chain is derived; the N-terminal segment of a heavy chain constant region (e.g., when it is linked to a heavy chain variable region via the N-terminus), the N-terminal segment of a light chain constant region (e.g., when it is linked to a light chain variable region via the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the N-terminal segment of a heavy chain constant region is ASTKGP (SEQ ID NO: 31). In some embodiments, the N-terminal segment of a light chain constant region is RTVAAP (SEQ ID NO: 30). In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)1, and (G4S)2.

[0109] In some embodiments, the PDL1 chain comprises a C-terminal linker at its C-terminus. In some embodiments, the C-terminal linker is selected from the group consisting of: the C-terminal contiguous segment of the PDL1 chain in the protein from which the PDL1 chain is derived; the C-terminal segment of a heavy chain constant region CH1 (e.g., when it is linked to a heavy chain variable region at the N-terminus), the C-terminal segment of a light chain constant region (e.g., when it is linked to a light chain variable region at the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the C-terminal segment of the PDL1 chain is selected from the group consisting of VKVNAPYNK (SEQ ID NO: 51), VKVNAPYNRGEC (SEQ ID NO: 52), VKVNAPYNKSC (SEQ ID NO: 53). In some embodiments, the C-terminal linker is selected from the group consisting of VKVNAPYNK, VKVNAPYNRGEC, VKVNAPYNKSC, GE, (G4S)1, and (G4S)2.

[0110] In some embodiments, the PDL1 chain comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at its N-terminus and a C-terminal linker VKVNAPYNK (SEQ ID NO: 51) or VKVNAPYNRGEC (SEQ ID NO: 52) at its C-terminus. In some embodiments, the PDL1 chain comprises an N-terminal linker ASTKGP (SEQ ID NO: 31) at its N-terminus and a C-terminal linker VKVNAPYNK (SEQ ID NO: 51) or VKVNAPYNKSC (SEQ ID NO: 53) at its C-terminus.

[0111] In some embodiments, the modified PDL1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 15-18, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 15-18. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51 or 52. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51 or 53.

[0112] In some embodiments, the modified PDL1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15 or 16, and comprises N17T, D104C. In some embodiments, the modified PDL1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or 16.

[0113] In some embodiments, the modified PDL1 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 17 or 18, and comprises N17T, T4A, K23A, Q73A. In some embodiments, the modified PDL1 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 or 18.

[0114] PDL2 chain

[0115] In some embodiments, the PDL2 chain is from the extracellular immunoglobulin variable region (IgV)-like domain of a PDL2 protein, which can bind to PD1. In some embodiments, the PDL2 chain is a wild-type IgV-like domain or a fragment of a wild-type IgV-like domain, e.g., it comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0116] In some embodiments, the PDL2 chain used in the polypeptide complex of the present disclosure is a modified PDL2 chain that has mutations compared to a wild-type IgV-like domain fragment, e.g., a parent PDL2 chain.

[0117] In some embodiments, the parent PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. When referring to the mutation positions of a modified PDL2 chain herein, the mutation positions are all relative to the sequential numbering positions of the sequence SEQ ID NO: 23, i.e., taking the first amino acid of SEQ ID NO: 23 as the first position, regardless of whether it further comprises linkers at the N-terminal and / or C-terminal.

[0118] In some embodiments, the modified PDL2 chain suitable for use in the polypeptide complex of the present disclosure has a cysteine mutation (i.e., mutated to cysteine) to generate an interchain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as D), preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or the binding of the PDL2 chain to the PD1 chain, compared to a wild-type IgV-like domain fragment such as SEQ ID NO: 23.

[0119] In some embodiments, the PDL2 chain has a mutation of one or more amino acid residues selected from positions 17, 44, or 88, wherein the PDL2 chain mutation positions are relative to the sequential numbering positions of the sequence SEQ ID NO: 23.

[0120] In one embodiment, the modified PDL2 chain has amino acid mutations in positions 17 and 44, and optionally one or more other amino acid mutations, e.g., a mutation at position 88, wherein the PDL2 chain mutation positions are sequentially numbered positions relative to sequence SEQ ID NO: 23.

[0121] In some embodiments, the amino acid residue at position 17 is mutated to D, the amino acid residue at position 44 is mutated to D and / or the amino acid residue at position 88 is mutated to C.

[0122] In some embodiments, the modified PDL2 chain has one or more amino acid residue mutations selected from 17D, 44D, or 88C, wherein the PDL2 chain mutation positions are sequentially numbered positions relative to sequence SEQ ID NO: 23.

[0123] In some embodiments, the modified PDL2 chain has amino acid residue mutations of 17D and 44D, and optionally one or more other amino acid mutations, e.g., 88C, wherein the PDL2 chain mutation positions are sequentially numbered positions relative to sequence SEQ ID NO: 23.

[0124] In some embodiments, the modified PDL2 chain has the following combinations of mutations:

[0125] N17D, N44D, V88C; or

[0126] N17D, N44D.

[0127] In some embodiments, the modified PDL2 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 28 or 29, or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or 29.

[0128] In some embodiments, the modified PDL2 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 28, and comprises N17D, N44D, V88C. In some embodiments, the modified PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.

[0129] In some embodiments, the modified PDL2 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 29, and comprises N17D and N44D. In some embodiments, the modified PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29.

[0130] In some embodiments, the PDL2 chain suitable for use in a polypeptide complex of the disclosure is a PDL2 chain comprising a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus, e.g., a modified PDL2 chain comprising a N-terminal and / or C-terminal linker.

[0131] In some embodiments, the PDL2 chain suitable for use in a polypeptide complex of the disclosure comprises a N-terminal linker at its N-terminus and is linked to a heavy chain variable region or a light chain variable region via the N-terminal linker. In some embodiments, the N-terminal linker is selected from the group consisting of: the N-terminal contiguous segment of the PDL2 chain in the protein from which the PDL2 chain is derived; the N-terminal segment of a heavy chain constant region (e.g., when it is linked to a heavy chain variable region via the N-terminus), the N-terminal segment of a light chain constant region (e.g., when it is linked to a light chain variable region via the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the N-terminal segment of a heavy chain constant region is ASTKGP (SEQ ID NO: 31). In some embodiments, the N-terminal segment of a light chain constant region is RTVAAP (SEQ ID NO: 30). In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2.

[0132] In some embodiments, the PDL2 chain comprises a C-terminal linker at the C-terminus. In some embodiments, the C-terminal linker is selected from the C-terminal contiguous segment of the PDL2 chain in the protein from which the PDL2 chain is derived; a C-terminal segment of a heavy chain constant region CHI (e.g., when it is N-terminally linked to a heavy chain variable region), a C-terminal segment of a light chain constant region (e.g., when it is N-terminally linked to a light chain variable region), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the C-terminal segment of the PDL2 chain is selected from VKASYRK (SEQ ID NO: 54), VKASYRKSC (SEQ ID NO: 55), or VKASYRKGEC (SEQ ID NO: 56). In some embodiments, the C-terminal linker is selected from VKASYRK (SEQ ID NO: 54), VKASYRKSC (SEQ ID NO: 55), or VKASYRKGEC (SEQ ID NO: 56), GE, (G4S)i, and (GS)2.

[0133] In some embodiments, the PDL2 chain comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) or VKASYRKGEC (SEQ ID NO: 56) at the C-terminus. In some embodiments, the PDL2 chain comprises an N-terminal linker ASTKGP (SEQ ID NO: 31) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) or VKASYRKSC (SEQ ID NO: 55) at the C-terminus.

[0134] In some embodiments, the modified PDL2 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 24-27, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 24-27. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54 or 56. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54 or 55.

[0135] In some embodiments, the modified PDL2 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24 or 25, and comprises N17D, N44D, V88C. In some embodiments, the modified PDL2 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or 25.

[0136] In some embodiments, the modified PDL2 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 26 or 27, and comprises N17D, N44D. In some embodiments, the modified PDL2 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26 or 27.

[0137] CD47 chain

[0138] In some embodiments, the CD47 chain is from the extracellular immunoglobulin variable region (IgV)-like domain of the CD47 protein, which can bind to SIRPa. In some embodiments, the CD47 chain is a wild-type IgV-like domain or is part of a fragment of a wild-type IgV-like domain, e.g., it comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0139] In some embodiments, the CD47 chain used in the polypeptide complex of the present disclosure is a modified CD47 chain that has mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parental CD47 chain.

[0140] In some embodiments, the parental CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32. When referring to the mutated positions of the modified CD47 chain herein, the mutated positions are all the ordinal number positions relative to the sequence SEQ ID NO: 32, i.e., taking the first amino acid corresponding to the first position of SEQ ID NO: 32 as the first position start, regardless of whether it also comprises a linker at the N-terminal and / or C-terminal.

[0141] In some embodiments, the modified CD47 chain suitable for use in the polypeptide complex of the present disclosure has a cysteine mutation (i.e., mutated to cysteine) that produces an interchain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as S, K, D, Q, or E), preferably the N-glycosylation site mutation does not affect its interchain disulfide bond formation and / or binding of the CD47 chain to the SIRPa chain, as compared to a wild-type IgV-like domain fragment such as SEQ ID NO: 32.

[0142] In some embodiments, the modified CD47 chain suitable for use in the polypeptide complex of the present disclosure has one or more amino acid residue mutations selected from positions 5, 16, 29, 32, 46, 55, 93, or 104, wherein the modified CD47 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 32, as compared to a wild-type CD47 fragment such as SEQ ID NO: 32.

[0143] In some embodiments, the modified CD47 chain comprises a glycosylation site mutation, e.g., N at position 5, 16, 32, 55, and / or 93 is mutated to a non-glycosylated amino acid such as S, K, D, Q, or E. In some embodiments, the modified CD47 chain comprises a mutation to cysteine at position 29, 46, and / or 104.

[0144] In some embodiments, the modified CD47 chain has amino acid residue mutations selected from positions 5, 16, 55, and 93, and optionally one or more amino acid residue mutations (e.g., amino acid residue mutations at positions 29, 32, 46, or 104), wherein the modified CD47 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 32.

[0145] In some embodiments, the modified CD47 chain has

[0146] amino acid residue mutations at positions 5, 16, 32, 55, 93, and 104,

[0147] amino acid residue mutations at positions 5, 16, 29, 32, 55, and 93, or

[0148] amino acid residue mutations at positions 5, 16, 32, 46, 55, and 93,

[0149] wherein the modified CD47 chain mutation positions are sequentially numbered positions relative to the sequence of SEQ ID NO: 32. In some embodiments, the parent CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0150] In some embodiments, the amino acid residue at position 5 is mutated to K, the amino acid residue at position 16 is mutated to D, the amino acid at position 29 is mutated to C, the amino acid residue at position 32 is mutated to S, the amino acid residue at position 46 is mutated to C, the amino acid residue at position 55 is mutated to Q, the amino acid residue at position 93 is mutated to E or K, and / or the amino acid at position 104 is mutated to C.

[0151] In some embodiments, the modified CD47 chain has one or more amino acid residue mutations selected from 5K, 16D, 29C, 32S, 46C, 55Q, 93E / K, and 104C, wherein the CD47 chain mutation site is the sequentially numbered site relative to sequence SEQ ID NO: 32. In some embodiments, the CD47 chain has a combination of mutations 5K, 16D, 32S, 55Q, and 93E / K, and optionally one or more other mutations, e.g., 29C, 46C, and / or 104C.

[0152] In some embodiments, the modified CD47 chain has a combination of mutations as follows:

[0153] N5K, N16D, N32S, N55Q, N93E, E104C;

[0154] N5K, N16D, N32S, N55Q, N93K, E104C;

[0155] N5K, N16D, E29C, N32S, N55Q, N93E;

[0156] N5K, N16D, E29C, N32S, N55Q, N93K;

[0157] N5K, N16D, N32S, D46C, N55Q, N93E; or

[0158] N5K, N16D, N32S, D46C, N55Q, N93K.

[0159] In some embodiments, a modified CD47 chain suitable for use in a polypeptide complex of the present disclosure comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-50, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 45-50.

[0160] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 45, and comprises N5K, N16D, N32S, N55Q, N93E, E104C. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45.

[0161] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 46, and comprises N5K, N16D, N32S, N55Q, N93K, E104C. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46.

[0162] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 47, and comprises N5K, N16D, E29C, N32S, N55Q, N93E. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47.

[0163] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 48, and comprises N5K, N16D, E29C, N32S, N55Q, N93K. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48.

[0164] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 49, and comprises N5K, N16D, N32S, D46C, N55Q, N93E. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49.

[0165] In some embodiments, the modified CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 50, and comprises N5K, N16D, N32S, D46C, N55Q, N93K. In some embodiments, the modified CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50.

[0166] In some embodiments, the CD47 chain suitable for use in a polypeptide complex of the present disclosure is a CD47 chain comprising an N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus, e.g., a modified CD47 chain comprising an N-terminal and / or C-terminal linker.

[0167] In some embodiments, the CD47 chain suitable for use in a polypeptide complex of the present disclosure comprises an N-terminal linker at its N-terminus and is linked to a heavy chain variable region or a light chain variable region via the N-terminal linker. In some embodiments, the N-terminal linker is selected from the group consisting of: the N-terminal contiguous fragment of the CD47 chain in the protein from which the CD47 chain is derived; the N-terminal fragment of a heavy chain constant region (e.g., when it is linked to a heavy chain variable region via the N-terminus), the N-terminal fragment of a light chain constant region (e.g., when it is linked to a light chain variable region via the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the N-terminal linker of the CD47 chain is (G4S)n, where n = 1, 2, or 3, e.g., (G4S)i (SEQ ID NO: 76) or (GS)2(SEQ ID NO: 77). In some embodiments, the N-terminal fragment of a heavy chain constant region is ASTKGP (SEQ ID NO: 31). In some embodiments, the N-terminal fragment of a light chain constant region is RTVAAP (SEQ ID NO: 30). In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2.

[0168] In some embodiments, the CD47 chain comprises a C-terminal linker at the C-terminus. In some embodiments, the C-terminal linker is selected from the C-terminal contiguous fragment of the CD47 chain in the protein from which the CD47 chain is derived; a C-terminal fragment of a heavy chain constant region CHI (e.g., when it is N-terminally linked to a heavy chain variable region), a C-terminal fragment of a light chain constant region (e.g., when it is N-terminally linked to a light chain variable region), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the C-terminal linker of the CD47 chain is AKEPKSA (SEQ ID NO: 67). In some embodiments, the C-terminal linker is selected from AKEPKSA, GE, (G4S)i, and (GS)2.

[0169] In some embodiments, the CD47 chain comprises an N-terminal linker (G4S)i (SEQ ID NO: 76) at the N-terminus and a C-terminal linker AKEPKSA (SEQ ID NO: 67) or GE at the C-terminus.

[0170] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 33-44, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 33-44. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67 or the amino acids GE.

[0171] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 33 or 39, and comprises N5K, N16D, N32S, N55Q, N93E, E104C. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 or 39.

[0172] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 34 or 40, and comprises N5K, N16D, N32S, N55Q, N93K, E104C. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 or 40.

[0173] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 35 or 41, and comprises N5K, N16D, E29C, N32S, N55Q, N93E. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 or 41.

[0174] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 36 or 42, and comprises N5K, N16D, E29C, N32S, N55Q, N93K. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 or 42.

[0175] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 37 or 43, and comprises N5K, N16D, N32S, D46C, N55Q, N93E. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37 or 43.

[0176] In some embodiments, the modified CD47 chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38 or 44, and comprises N5K, N16D, N32S, D46C, N55Q, N93K. In some embodiments, the CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38 or 44.

[0177] SIRPa chain

[0178] In some embodiments, the SIRPa chain is from the extracellular immunoglobulin variable region (IgV)-like domain of a SIRPa protein, which can bind to CD47. In some embodiments, the SIRPa chain is a wild-type IgV-like domain or is part of a fragment of a wild-type IgV-like domain, e.g., it comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0179] In some embodiments, the SIRPa chain used in the polypeptide complex of the present disclosure is a modified SIRPa chain that has mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parent SIRPa chain.

[0180] In some embodiments, the parent SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57. When referring to the mutated positions of a modified SIRPa chain herein, the mutated positions are all sequentially numbered positions relative to the sequence of SEQ ID NO: 57, i.e., with the first amino acid corresponding to the first position of SEQ ID NO: 57 as the first position, regardless of whether it also comprises linkers at the N-terminal and / or C-terminal end.

[0181] In some embodiments, the modified SIRPa chain suitable for use in the polypeptide complex of the present disclosure has a cysteine mutation (i.e., mutated to cysteine) to generate an interchain disulfide bond and / or an N-glycosylation site mutation (i.e., mutating an N-glycosylation site to a non-glycosylated amino acid) compared to a wild-type fragment, e.g., SEQ ID NO: 57, preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or binding of the SIRPa chain to the CD47 chain.

[0182] In some embodiments, the modified SIRPa chain suitable for use in the polypeptide complex of the present disclosure has a mutation of an amino acid residue in one or more of positions 53, 69, or 98, wherein the modified SIRPa chain mutation position is sequentially numbered with respect to sequence SEQ ID NO: 57, as compared to a wild-type IgV-like domain fragment, e.g., SEQ ID NO: 57.

[0183] In some embodiments, the modified SIRPa chain comprises a mutation to cysteine at position 53, 69, and / or 98.

[0184] In some embodiments, the modified SIRPa chain has a mutation of an amino acid residue in one or more of positions 53, 69, or 98, wherein the modified SIRPa chain mutation position is sequentially numbered with respect to sequence SEQ ID NO: 57.

[0185] In some embodiments, the modified SIRPa chain has

[0186] a mutation of an amino acid residue in position 53,

[0187] a mutation of an amino acid residue in position 69, or

[0188] a mutation of an amino acid residue in position 98,

[0189] wherein the modified SIRPa chain mutation position is sequentially numbered with respect to sequence SEQ ID NO: 57.

[0190] In some embodiments, the parent SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0191] In some embodiments, the amino acid residue mutation at position 53, 69, or 98 is C.

[0192] In some embodiments, the modified SIRPa chain has one or more of the following mutations:

[0193] In some embodiments, the modified SIRPa chain has a mutation of:

[0194] K53C;

[0195] R69C; or

[0196] S98C.

[0197] In some embodiments, the modified SIRPa chain suitable for use in a polypeptide complex of the disclosure comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 64-66, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 64-66.

[0198] In some embodiments, the modified SIRPa chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 64, and comprises K53C. In some embodiments, the modified SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64.

[0199] In some embodiments, the modified SIRPa chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 65, and comprises R69C. In some embodiments, the modified SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

[0200] In some embodiments, the modified SIRPa chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 66, and comprises S98C. In some embodiments, the modified SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66.

[0201] In some embodiments, the SIRPa chain suitable for use in a polypeptide complex of the disclosure is a SIRPa chain comprising a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus, e.g., a modified SIRPa chain comprising a N-terminal and / or C-terminal linker.

[0202] In some embodiments, the SIRPa chain of the polypeptide complex useful in the present disclosure comprises a N-terminal linker at its N-terminus and is linked to the heavy chain variable region or the light chain variable region via the N-terminal linker. In some embodiments, the N-terminal linker is selected from the group consisting of: the N-terminal contiguous segment of the SIRPa chain in the protein from which the SIRPa chain is derived; the N-terminal segment of the heavy chain constant region (e.g., when it is linked to the heavy chain variable region via the N-terminus), the N-terminal segment of the light chain constant region (e.g., when it is linked to the light chain variable region via the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the N-terminal segment of the heavy chain constant region is ASTKGP (SEQ ID NO: 31). In some embodiments, the N-terminal segment of the light chain constant region is RTVAAP (SEQ ID NO: 30). In some embodiments, the N-terminal linker of the SIRPa chain is (G4S)n, where n = 1-10, e.g., (G4S)i (SEQ ID NO: 76) or (GS)2(SEQ ID NO: 77). In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2.

[0203] In some embodiments, the SIRPa chain comprises a C-terminal linker at its C-terminus. In some embodiments, the C-terminal linker is selected from the group consisting of: the C-terminal contiguous segment of the SIRPa chain in the protein from which the SIRPa chain is derived; the C-terminal segment of the heavy chain constant region CHI (e.g., when it is linked to the heavy chain variable region at the N-terminus), the C-terminal segment of the light chain constant region (e.g., when it is linked to the light chain variable region at the N-terminus), or (G4S)n, where n = 1-10, e.g., 1, 2, or 3. In some embodiments, the C-terminal linker of the SIRPa chain is EPKSA (SEQ ID NO: 68). In some embodiments, the C-terminal linker is selected from the group consisting of EPKSA, GE, (G4S)i, and (GS)2.

[0204] In some embodiments, the SIRPa chain comprises a N-terminal linker (G4S)i (SEQ ID NO: 76) at its N-terminus and a C-terminal linker EPKSA (SEQ ID NO: 68) or GE at its C-terminus.

[0205] In some embodiments, the modified SIRPa chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 58-63, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 58-63. In some embodiments, the N-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76, and / or the C-terminal linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 68, or the amino acids GE.

[0206] In some embodiments, the modified SIRPa chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 58 or 61, and comprises K53C. In some embodiments, the SIRPa chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or 61.

[0207] In some embodiments, the modified SIRPa chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 59 or 62, and comprises R69C. In some embodiments, the SIRPa chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or 62.

[0208] In some embodiments, the modified SIRPa chain comprising N-terminal and C-terminal linkers comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 60 or 63, and comprises S98C. In some embodiments, the SIRPa chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or 63.

[0209] Combination of the first domain and the second domain

[0210] The first domain and the second domain of the present disclosure are capable of interacting to form a dimer.

[0211] In some embodiments, the first or second domain comprises or consists of a PD1 chain comprising a N-terminal linker at its N-terminal end and a C-terminal linker at its C-terminal end. In some embodiments, the N-terminal linker is selected from the group consisting of DRPWNP, RTVAAP, ASTKGP, (G4S)i, and (G4S)2. In some embodiments, the C-terminal linker is selected from the group consisting of ERRAE, ERRAESC, ERRGEC, GE, (G4S)i, and (GS)2. In some embodiments, the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the PD1 chain has mutations compared to the fragment of the wild-type IgV-like domain, e.g. the parental PD1 chain. In some embodiments, the PD1 chain has cysteine mutations and / or N-glycosylation site mutations compared to the fragment of the wild-type IgV-like domain, e.g. the parental PD1 chain. In some embodiments, the PD1 chain has a combination of mutations N15S, N24S, N40S, C59S, N82D, and optionally one or more other mutations, e.g. 30C, and / or 44T / C. In some preferred embodiments, the PD1 chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 8-11, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 8-11. In some embodiments, the first or second domain comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 2-7.

[0212] In some embodiments, the first or second domain comprises or consists of a PDL1 chain comprising a N-terminal linker at its N-terminus and a C-terminal linker at its C- terminus. In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2. In some embodiments, the C-terminal linker is selected from the group consisting of VKVNAPYNK, VKVNAPYNRGEC, VKVNAPYNKSC, GE, (G4S)i, and (GS)2. In some embodiments, the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the PDL1 chain has mutations compared to a fragment of a wild-type IgV-like domain (e.g., a parent PDL1 chain). In some embodiments, the PDL1 chain has a cysteine mutation (i.e., mutated to cysteine) that creates an inter-chain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as T), compared to a fragment of a wild-type IgV-like domain (e.g., a parent PDL1 chain). In some embodiments, the PDL1 chain has an amino acid residue mutation of 17T, and optionally one or more other amino acid mutations, such as 104C, 4A, 23A, or 73A. In some preferred embodiments, the PDL1 chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 19 or 20, or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 20. In some embodiments, the first or second domain comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 15-18.

[0213] In some embodiments, the first or second domain comprises or consists of a PDL2 chain comprising an N-terminal linker at its N-terminus and a C-terminal linker at its C- terminus. In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2. In some embodiments, the C-terminal linker is selected from the group consisting of VKASYRK (SEQ ID NO: 54), VKASYRKSC (SEQ ID NO: 55), or VKASYRKGEC (SEQ ID NO: 56), GE, (G4S)i, and (GS)2. In some embodiments, the PDL2 chain comprises or is a fragment of a wild-type IgV-like domain, e.g., comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the PDL2 chain has mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parent PDL2 chain. In some embodiments, the PDL2 chain has a cysteine mutation (i.e., mutated to cysteine) to create an interchain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as D) compared to a fragment of a wild-type IgV-like domain, e.g., a parent PDL1 chain. In some embodiments, the PDL2 chain has amino acid residue mutations of 17D and 44D, and optionally one or more other amino acid mutations, e.g., 88C. In some preferred embodiments, the PDL2 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 28 or 29, or comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or 29. In some embodiments, the first or second domain comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 24-27.

[0214] In some embodiments, the first or second domain comprises or consists of a CD47 chain comprising a N-terminal linker at its N-terminus and a C-terminal linker at its C- terminus. In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2. In some embodiments, the C-terminal linker is selected from the group consisting of AKEPKSA, GE, (G4S)i, and (GS)2. In some embodiments, the CD47 chain comprises or is part of a fragment of a wild-type IgV-like domain, e.g., it comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the CD47 chain has mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parental CD47 chain. In some embodiments, the CD47 chain has mutations compared to a fragment of a wild-type IgV-like domain, e.g., a parental CD47 chain, that create inter-chain disulfide bonds (i.e., mutations to cysteine), and / or N-glycosylation site mutations (i.e., mutations of N-glycosylation sites to non-glycosylated amino acids such as S, K, D, Q, or E). In some embodiments, the CD47 chain has a combination of mutations of 5K, 16D, 32S, 55Q, and 93E / K, and optionally one or more other mutations, e.g., 29C, 46C, and / or 104C. In some preferred embodiments, the CD47 chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-50, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 45-50. In some embodiments, the first or second domain comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 33-44.

[0215] In some embodiments, the first or second domain comprises or consists of a SIRPa chain comprising a N-terminal linker at its N-terminus and a C-terminal linker at its C- terminus. In some embodiments, the N-terminal linker is selected from the group consisting of RTVAAP, ASTKGP, (G4S)i, and (G4S)2. In some embodiments, the C-terminal linker is selected from the group consisting of EPKSA, GE, (G4S)i, and (GS)2. In some embodiments, the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the SIRPa chain has mutations compared to a fragment of the wild-type IgV-like domain (e.g., a parent SIRPa chain). In some embodiments, the SIRPa chain has a cysteine mutation (i.e., mutated to cysteine) that creates an interchain disulfide bond, and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid) compared to a fragment of the wild-type IgV-like domain (e.g., a parent SIRPa chain). In some embodiments, the SIRPa chain has one or more of the amino acid residues 53C, 69C, and / or 98C mutated. In some preferred embodiments, the SIRPa chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 64-66, or comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 64-66. In some embodiments, the first or second domain comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 58-63.

[0216] Thus, in some embodiments, the present disclosure provides combinations of first and second domains as shown in Table A below, optionally the first and second domains in Table A can be interchanged:

[0217] Table A: Exemplary combinations of first and second domains

[0218] In some embodiments, the present disclosure provides combinations of first and second domains as shown in Table B below:

[0219] Table B: Exemplary combinations of first and second domains:

[0220] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.

[0221] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 8, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0222] In some embodiments, the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0223] In some embodiments, the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 19, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0224] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0225] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0226] In some embodiments, the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0227] In some embodiments, the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0228] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

[0229] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 10, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0230] In some embodiments, the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0231] In some embodiments, the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 20, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0232] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27.

[0233] In some embodiments, the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 11, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0234] In some embodiments, the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0235] In some embodiments, the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 29, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0236] In some embodiments, the present disclosure provides combinations of first and second domains as follows in Table C, optionally the first and second domains in Table A can be interchanged:

[0237] Table C: Exemplary combinations of first and second domains

[0238] In some embodiments, the present disclosure provides combinations of first and second domains as follows in Table D:

[0239] Table D: Exemplary combinations of first and second domains

[0240] In some embodiments, the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0241] In some embodiments, the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 45 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0242] In some embodiments, the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0243] In some embodiments, the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 46 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0244] In some embodiments, the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59.

[0245] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 48 and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 65.

[0246] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 36 or consists of thereof, and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 59 or consists of thereof.

[0247] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 48 and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 65.

[0248] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 37 or consists of thereof, and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 60 or consists of thereof.

[0249] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 49 and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 66.

[0250] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 38 or consists of thereof, and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 60 or consists of thereof.

[0251] In some embodiments, the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 50 and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 66.

[0252] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises an amino acid sequence set forth in SEQ ID NO: 61 or consists of thereof, and the CD47 chain comprises an amino acid sequence set forth in SEQ ID NO: 39 or consists of thereof.

[0253] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0254] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0255] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0256] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0257] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0258] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0259] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0260] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44.

[0261] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0262] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44.

[0263] In some embodiments, the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0264] II. Dimerization Domain

[0265] In some embodiments, the first dimerization domain is a first Fc region, and the second dimerization domain is a second Fc region. In some embodiments, the first Fc region and the second Fc region are the same or different.

[0266] In some embodiments, the first Fc region and the second Fc region are the same, which are capable of dimerization to form a homodimer Fc scaffold. In some embodiments, when the polypeptide complex of the present disclosure comprises two first antigen binding moieties, the Fc regions to which the antigen binding moieties are attached are also the same.

[0267] In some embodiments, the first Fc region and the second Fc region are different, which are capable of dimerization to form a heterodimer Fc scaffold.

[0268] In this context, an Fc region refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and can include native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass intact human Fc regions of the various immunoglobulin subtypes (e.g., IgG, IgA, IgE, IgM, etc.) and subtypes (e.g., IgG1, IgG2, IgG3, IgG4, etc.) as well as variants thereof.

[0269] In some embodiments, the Fc region of the present disclosure comprises antibody CH2 and CH3.

[0270] In some embodiments, the antibody Fc region can also carry an IgG hinge region or a partial IgG hinge region at the N-terminus, e.g., an IgG1 hinge region or a partial IgG1 hinge region, e.g., the sequence from D221 to P230 according to the EU numbering. Mutations can be contained in the hinge region.

[0271] Unless otherwise indicated herein, numbering of amino acid residues in the Fc region is in accordance with the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0272] In some embodiments, the first Fc region and the second Fc region are derived from IgG1, IgG2, IgG3 or IgG4, e.g., are Fc regions of human IgG1, IgG2, IgG3 or IgG4.

[0273] In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or more thereto.

[0274] As understood by the skilled person, in order to facilitate the formation of the polypeptide complex of the disclosure as a heterodimer, the Fc region comprised by the polypeptide complex of the disclosure can comprise mutations that facilitate heterodimerization of the first Fc region with the second Fc region. In one embodiment, mutations are introduced in the CH3 region of both Fc regions.

[0275] Methods to promote heterodimerization of Fc regions are known in the art. For example, the CH3 regions of a first Fc region and a second Fc region are engineered in a complementary fashion such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the other, complementary engineered CH3 region (such that the CH3 regions of the first and second Fc region heterodimerize and no homodimers between two first CH3 regions or two second CH3 regions are formed). Exemplary techniques include, e.g., knobs-into-holes (Ridgway et al., Protein Engineering, 9(7), pp. 617-21 (1996); Merchant et al., Nature Biotechnology, 16(7), pp. 677-681 (1998)), electrostatics (Gunasekaran et al., Journal of Biological Chemistry, 285(25), pp. 19637-19646 (2010)), or negative stater design (Kreudenstein et al., mAbs, 5(5), pp. 646-654 (2013); Leaver-Fay et al., Structure, 24(4), pp. 641-651 (2016)).

[0276] Preferably, based on the Knob-in-Hole technology, a respective Knob mutation and Hole mutation are introduced in the first Fc region and the second Fc region. This technology is described, e.g., in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996), Merchant et al., Nature Biotechnology, 16(7), pp. 677-681 (1998), and Carter, J Immunol Meth 248, 7-15 (2001). In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0277] In a particular embodiment, one Fc region comprises the amino acid substitution T366W, and the other Fc region comprises the amino acid substitutions T366S, L368A, and Y407V (numbering according to the EU index).

[0278] In a particular embodiment, the Fc region can further comprise mutations for improved purification, e.g., mutations at position H445 and / or Y446 to reduce the affinity to Protein A. Thus, the Fc region can comprise the H445R and Y446F mutations.

[0279] Thus, in a particular embodiment, the multispecific polypeptide complex of the present disclosure comprises two Fc regions heterodimerized, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78 and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79.

[0280] Thus, in a particular embodiment, the multispecific polypeptide complex of the present disclosure comprises two Fc regions heterodimerized, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78 and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79.

[0281] Thus, in a particular embodiment, the multispecific polypeptide complex of the present disclosure comprises two Fc regions heterodimerized, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78 and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79.

[0282] The Fc region in the polypeptide complex, e.g., antibody, of the present disclosure can also be mutated to obtain desired properties. As will be appreciated by those skilled in the art, depending on the intended use of the polypeptide complex of the present disclosure, the polypeptide complex of the present disclosure can also comprise modifications in the Fc domain that alter binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcy receptor, particularly a human Fcy receptor. In some embodiments, the Fc region comprises mutations that reduce binding to Fcy receptors. For example, in some embodiments, the Fc region used in the present disclosure has L234A / L235A mutations that reduce binding to Fcy receptors. In yet another preferred embodiment, the Fc fragment can have mutations that result in increased serum half-life, e.g., mutations that improve the binding of the Fc fragment to FcRn. In some embodiments, the Fc region is modified with respect to properties of effector function of the Fc region, e.g., complement activation function of the Fc region. In some embodiments, the effector function has been reduced or eliminated relative to a wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modifications. In some preferred embodiments, the Fc region has reduced effector function mediated by the Fc region, e.g., reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprising mutations that achieve the above functions.

[0283] III. Antigens and Antigen Binding Sites

[0284] The polypeptide complex of the present disclosure, e.g., monospecific or multispecific antibody such as bispecific antibody, can bind to any target, e.g., antigen.

[0285] In some embodiments, the antigen is a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the tumor-associated antigen is an immune checkpoint molecule.

[0286] In some embodiments, the antigen is selected from C-Met, EGFR, HER3, 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1 lc, CD123, CD138, CD142, CD147, CD166, CD19, CD19, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200, CD200CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45 (PTPRC), CD46, CD47, CD49D (ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-a, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PDL1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, short proteoglycans, mesothelin, sodium phosphate cotransporter 2B, Claudin 18.2, endoglin receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin a4b7, integrin avb6, trophoblast glycoprotein, and tissue factor.

[0287] In some embodiments, the polypeptide complex specifically binds one or more antigens disclosed herein, for example specifically binds two antigens disclosed herein. In some embodiments, the polypeptide complex specifically binds C-Met, EGFR, HER3 or PDL1.

[0288] In some embodiments, the polypeptide complex specifically binds EGFR and HER3, or HER3 and C-Met, or HER3 and PDL1.

[0289] The VH and VL of an antigen binding moiety of the present disclosure can constitute an antigen binding site. The VH and VL of an antigen binding site of the present disclosure can be derived from an antibody that binds to an antigen described above. In some embodiments, the antigen binding site of the present disclosure is selected from the antigen binding sites shown below.

[0290] Antigen binding site specifically binding C-met

[0291] In some embodiments, the antigen binding site is derived from an antibody that specifically binds C-met, for example an antibody that specifically binds C-met disclosed in WO2005016382A1, for example CE-355621 disclosed therein.

[0292] In some embodiments, the antigen binding site specifically binding C-met comprises the 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH and the 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region VL of an antibody that specifically binds C-met, for example an antibody that specifically binds C-met disclosed in WO2005016382A1, for example CE-355621 disclosed therein, wherein said HCDR1, HCDR2 and HCDR3 and said LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, for example Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably said HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0293] In some embodiments, the antigen binding site specifically binding C-met comprises the heavy chain variable region VH and the light chain variable region VL of an antibody that specifically binds C-met, for example an antibody that specifically binds C-met disclosed in WO2005016382A1, for example CE-355621 disclosed therein.

[0294] In some embodiments, the antigen binding site that specifically binds C-met comprises a heavy chain variable region VH which comprises 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as depicted in SEQ ID NO: 83, wherein the HCDR1, HCDR2 and HCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 are defined according to the Kabat definition rule.

[0295] In some embodiments, the antigen binding site that specifically binds C-met comprises a light chain variable region VL which comprises 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as depicted in SEQ ID NO: 84, wherein the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0296] In some embodiments, the antigen binding site that specifically binds C-met comprises a VH and a VL, wherein the VH comprises 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as depicted in SEQ ID NO: 83, and the VL comprises 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as depicted in SEQ ID NO: 84, wherein the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0297] In some embodiments, the antigen binding site that specifically binds C-met comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3, and the VL comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 144; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 145, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 146, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 147, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 148, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 149.

[0298] In some aspects, the antigen binding site that specifically binds C-met comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH

[0299] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83; or

[0300] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 83; and / or

[0301] the VL

[0302] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84; or

[0303] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 84.

[0304] In some embodiments, the antigen binding site that specifically binds C-met comprises a VH and a VL, wherein the VH comprises the sequence set forth in SEQ ID NO: 83 or consists of said sequence; and the VL comprises the sequence set forth in SEQ ID NO: 84 or consists of said sequence.

[0305] antigen binding site that specifically binds EGFR

[0306] In some embodiments, the antigen binding site is derived from an antibody that specifically binds to EGFR, such as an antibody that specifically binds to EGFR disclosed in WO2002100348A2, such as Zalutumumab disclosed therein.

[0307] In some embodiments, the antigen binding site that specifically binds to EGFR comprises 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region VH and 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region VL of an antibody that specifically binds to EGFR, such as an antibody that specifically binds to EGFR disclosed in WO2002100348A2, such as Zalutumumab disclosed therein, wherein said HCDR1, HCDR2 and HCDR3 and said LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably said HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0308] In some embodiments, the antigen binding site that specifically binds to EGFR comprises a heavy chain variable region VH and a light chain variable region VL of an antibody that specifically binds to EGFR, such as an antibody that specifically binds to EGFR disclosed in WO2002100348A2, such as Zalutumumab disclosed therein.

[0309] In some embodiments, the antigen binding site that specifically binds to EGFR comprises a heavy chain variable region VH comprising 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as depicted in SEQ ID NO: 85, wherein said HCDR1, HCDR2 and HCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably said HCDR1, HCDR2 and HCDR3 are defined according to the Kabat definition rule.

[0310] In some embodiments, the antigen binding site that specifically binds to EGFR comprises a light chain variable region VL comprising 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as depicted in SEQ ID NO: 86, wherein said LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably said LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0311] In some embodiments, the antigen-binding site that specifically binds EGFR comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 150; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 151, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 152, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 153, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 154, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 155.

[0312] In some embodiments, the antigen-binding site that specifically binds EGFR comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 150; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 151, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 152, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 153, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 154, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 155.

[0313] In some aspects, the antigen-binding site that specifically binds EGFR comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH

[0314] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 85; or

[0315] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 85; and / or

[0316] the VL

[0317] (i) comprises or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 86; or

[0318] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 86.

[0319] In some embodiments, the antigen binding site that specifically binds to EGFR comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 85; and the VL comprises or consists of the sequence set forth in SEQ ID NO: 86.

[0320] antigen binding site that specifically binds to HER3

[0321] In some embodiments, the antigen binding site is derived from an antibody that specifically binds to HER3, e.g., an antibody that specifically binds to HER3 disclosed in WO2022262772A1, e.g., Clone-2 therein.

[0322] In some embodiments, the antigen binding site that specifically binds to HER3 comprises the 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region VH and the 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region VL of an antibody that specifically binds to HER3, e.g., an antibody that specifically binds to HER3 disclosed in WO2022262772A1, e.g., Clone-2 therein, wherein the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, e.g., Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0323] In some embodiments, the antigen binding site that specifically binds to HER3 comprises a heavy chain variable region VH and a light chain variable region VL of an antibody that specifically binds to HER3, e.g., an antibody that specifically binds to HER3 disclosed in WO2022262772A1, e.g., Clone-2 therein.

[0324] In some embodiments, the antigen binding site that specifically binds HER3 comprises a heavy chain variable region VH which comprises 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as depicted in SEQ ID NO: 87, wherein the HCDR1, HCDR2 and HCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 are defined according to the Kabat definition rule.

[0325] In some embodiments, the antigen binding site that specifically binds HER3 comprises a light chain variable region VL which comprises 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as depicted in SEQ ID NO: 88, wherein the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0326] In some embodiments, the antigen binding site that specifically binds HER3 comprises a VH and a VL, wherein the VH comprises 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as depicted in SEQ ID NO: 87, and the VL comprises 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as depicted in SEQ ID NO: 88, wherein the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, such as Kabat, AbM, Chothia, or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0327] In some embodiments, the antigen binding site that specifically binds HER3 comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3, and the VL comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 156; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 157, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 158, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 159, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 160, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 161.

[0328] In some aspects, the antigen binding site that specifically binds HER3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH

[0329] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 87; or

[0330] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 87; and / or

[0331] the VL

[0332] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88; or

[0333] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 88.

[0334] In some embodiments, the antigen binding site that specifically binds HER3 comprises a VH and a VL, wherein the VH comprises the sequence set forth in SEQ ID NO: 87 or consists of said sequence; and the VL comprises the sequence set forth in SEQ ID NO: 88 or consists of said sequence.

[0335] antigen binding site that specifically binds PDL1

[0336] In some embodiments, the antigen binding site is derived from an antibody that specifically binds PDL1, e.g., an antibody that specifically binds PDL1 disclosed in WO2021036929A1, e.g., Clone-2 therein.

[0337] In some embodiments, the antigen binding site that specifically binds PDL1 comprises 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region VH and 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region VL of an antibody that specifically binds PDL1, e.g., Clone-2 in WO2021036929A1, wherein the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, e.g., Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0338] In some embodiments, the antigen binding site that specifically binds PDL1 comprises a heavy chain variable region VH and a light chain variable region VL of an antibody that specifically binds PDL1, e.g., Clone-2 in WO2021036929A1.

[0339] In some embodiments, the antigen binding site that specifically binds PDL1 comprises a heavy chain variable region VH comprising 3 HCDR1, HCDR2 and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 162, wherein the HCDR1, HCDR2 and HCDR3 can be defined according to any definition rule, e.g., Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably the HCDR1, HCDR2 and HCDR3 are defined according to the Kabat definition rule.

[0340] In some embodiments, the antigen binding site that specifically binds PDL1 comprises a light chain variable region VL comprising 3 LCDR1, LCDR2 and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 163, wherein the LCDR1, LCDR2 and LCDR3 can be defined according to any definition rule, e.g., Kabat, AbM, Chothia or IMGT, or a combination thereof, preferably the LCDR1, LCDR2 and LCDR3 are defined according to the Kabat definition rule.

[0341] In some embodiments, the antigen binding site that specifically binds to PDL1 comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 164; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 165, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 166, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 167, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 168, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 169.

[0342] In some embodiments, the antigen binding site that specifically binds to PDL1 comprises a VH and a VL, wherein the VH comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 164; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 165, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 166, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 167, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 168, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 169.

[0343] In some aspects, the antigen binding site that specifically binds to PDL1 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH

[0344] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 162; or

[0345] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 162; and / or

[0346] the VL

[0347] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 163; or

[0348] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 163.

[0349] In some embodiments, the antigen binding site that specifically binds PDL1 comprises a VH and a VL, wherein the VH comprises or consists of the sequence set forth in SEQ ID NO: 162; and the VL comprises or consists of the sequence set forth in SEQ ID NO: 163.

[0350] IV. Monospecific antibodies

[0351] In some embodiments, the polypeptide complex of the present disclosure is a monospecific antibody. In some embodiments, the polypeptide antibody of the present disclosure is a full-length antibody, wherein the CH1 of both half antibodies is replaced by the first domain and the two CL are replaced by the second domain.

[0352] In some embodiments, the polypeptide complex of the present disclosure is a monospecific antibody comprising or consisting of two antigen binding moieties, wherein each antigen binding moiety specifically binds to an antigen and comprises or consists of:

[0353] a first polypeptide chain or heavy chain comprising or consisting of, from N- to C- terminus, a heavy chain variable region VH - a first domain (Domain 1) - a Fc region, or a heavy chain variable region VH - a first domain (Domain 1) - a Fc region;

[0354] a second polypeptide chain or light chain comprising or consisting of, from N- to C- terminus, a light chain variable region VL - a second domain (Domain 2), or a light chain variable region VL - a second domain (Domain 2);

[0355] wherein the first domain (Domain 1) and the second domain (Domain 2) are capable of interacting to form a dimer.

[0356] In some embodiments, the Fc region is or is from an IgG Fc, e.g., is or is from the Fc region of (human) IgGl, IgG2, IgG3, or IgG4, e.g., the Fc region of wild-type human IgGl.

[0357] In some embodiments, the first domain comprises an N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus. In some embodiments, the second domain comprises an N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus.

[0358] In some embodiments, the first domain comprises a N-terminal linker at its N-terminus and a C-terminal linker at its C-terminus and the second domain comprises a N-terminal linker at its N-terminus and a C-terminal linker at its C-terminus.

[0359] In some embodiments, the first domain and the second domain, or the first domain comprising a N-terminal linker and a C-terminal linker and the second domain comprising a N-terminal linker and a C-terminal linker, are a first domain and a second domain as defined herein above. For example, the first domain and the second domain are a combination of a first domain and a second domain as defined in Part I of the present disclosure “Combination of a first domain and a second domain”, e.g. a combination of Table A, Table B, Table C and Table D, respectively.

[0360] In some embodiments, the heavy chain variable region VH and the light chain variable region VL constitute an antigen binding site that specifically binds to an antigen. In some embodiments, the heavy chain variable region VH and the light chain variable region VL are a VH and a VL as defined herein above.

[0361] In some embodiments, the monospecific antibody of the present disclosure specifically binds to C-met, comprising a heavy chain and a light chain, wherein the VH is a VH of an antigen binding site that specifically binds to C-met as defined in Part III of the present disclosure, and the VL is a VL of an antigen binding site that specifically binds to C-met as defined in Part III of the present disclosure, and the first domain and the second domain are a combination of a first domain and a second domain as defined in Part I of the present disclosure “Combination of a first domain and a second domain”, e.g. a combination of Table A, Table B, Table C and Table D.

[0362] In some embodiments, the monospecific antibody of the present disclosure specifically binds to C-met, comprising a heavy chain and a light chain, wherein

[0363] a) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 89, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 90;

[0364] b) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 91 and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 92;

[0365] c) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 93 and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 94;

[0366] d) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 95 and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 96;

[0367] e) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 97 and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 98;

[0368] f) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 99 and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100;

[0369] g) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 101, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102;

[0370] h) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 103, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 104;

[0371] i) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 105, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 106;

[0372] j) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 107, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 108;

[0373] k) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 109, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 110;

[0374] l) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 111, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 112;

[0375] m) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 113, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 114;

[0376] n) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 115, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 116;

[0377] o) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 117, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 118;

[0378] p) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 119, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 120;

[0379] q) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 121, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 122;

[0380] r) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 123, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 124;

[0381] s) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 125, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 126; or

[0382] t) the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 127, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 128.

[0383] In some embodiments, the monospecific antibody of the present disclosure specifically binds C-met, comprising a heavy chain and a light chain, wherein

[0384] a) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90;

[0385] b) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 91 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 92;

[0386] c) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 93 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 94;

[0387] d) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96;

[0388] e) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98;

[0389] f) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 99 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 100;

[0390] g) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 102;

[0391] h) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 104;

[0392] i) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 105 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 106;

[0393] j) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 107 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 108;

[0394] k) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 109 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 110;

[0395] l) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 111, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 112;

[0396] m) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 113, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 114;

[0397] n) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 115, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 116;

[0398] o) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 117, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 118;

[0399] p) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 119, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 120;

[0400] q) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 121, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 122;

[0401] r) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 123, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 124;

[0402] s) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 125, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 126; or

[0403] t) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 127, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 128.

[0404] In some embodiments, the monospecific antibody of the present disclosure specifically binds HER3, comprising a heavy chain and a light chain, wherein the VH is a VH of an antigen binding site that specifically binds HER3 as defined in Section III herein, and the VL is a VL of an antigen binding site that specifically binds HER3 as defined in Section III herein, and the first domain and the second domain are a combination of a first domain and a second domain as defined in Section I herein "Combination of first domain and second domain", e.g. a combination of Table A, Table B, Table C and Table D, e.g. a combination of group 3 of Table A or Table B;

[0405] e.g. the first domain is a PD1 chain and the second domain is a PDL2 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24; or

[0406] the first domain is a PD1 chain and the second domain is a PDL2 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0407] In some embodiments, the monospecific antibody of the present disclosure specifically binds HER3, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 129, and the light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the monospecific antibody of the present disclosure specifically binds HER3, comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 129, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 130.

[0408] V. Bispecific antibodies

[0409] In some embodiments, the polypeptide complex of the present disclosure is a multispecific antibody, e.g. a bispecific antibody, comprising a first antigen binding moiety and one or more further antigen binding moieties. In some embodiments, the polypeptide complex of the present disclosure is a bispecific antibody, comprising or consisting of a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety comprises or consists of:

[0410] a first polypeptide chain or first heavy chain comprising from N-terminal to C-terminal a heavy chain variable region VH1 - a first domain (Domain 1 ) - a first Fc region, or a heavy chain variable region VH1 - a first domain (Domain 1 ) - a first Fc region;

[0411] a second polypeptide chain or first light chain comprising from N-terminal to C-terminal a light chain variable region VL1 - a second domain (Domain 2), or a light chain variable region VL1 - a second domain (Domain 2);

[0412] and the second antigen binding moiety comprises:

[0413] a third polypeptide chain or second heavy chain comprising from N-terminal to C-terminal a heavy chain variable region VH2 - CH1 - a second Fc region, or a heavy chain variable region VH2 - CH1 - a second Fc region;

[0414] a fourth polypeptide chain or second light chain comprising from N-terminal to C-terminal a light chain variable region VL2 - CL, or a light chain variable region VL2 - CL;

[0415] wherein the first antigen binding moiety specifically binds to a first antigen and the second antigen binding moiety specifically binds to a second antigen, the first domain (Domain 1 ) and the second domain (Domain 2) are capable of interacting to form a dimer.

[0416] In some embodiments, the Fc region is or is from an IgG Fc, e.g. from an Fc region of (human) IgG1, IgG2, IgG3 or IgG4. In some embodiments, the first Fc region and the second Fc region are different, e.g. they comprise Knob-into-hole mutations, respectively. In some embodiments, the first Fc region comprises a Knob mutation, e.g. T366W, and the second Fc region comprises a Hole mutation, e.g. T366S, L368A and Y407V, and optionally H445R and Y446F. In some embodiments, the first Fc region and the second Fc region are Fc regions as defined herein above.

[0417] In some embodiments, the first domain comprises a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus. In some embodiments, the second domain comprises a N-terminal linker at its N-terminus and / or a C-terminal linker at its C-terminus. In some embodiments, the first domain comprises a N-terminal linker at its N-terminus and a C-terminal linker at its C-terminus and the second domain comprises a N-terminal linker at its N-terminus and a C-terminal linker at its C-terminus. In some embodiments, the first domain and the second domain, the first domain comprising a N-terminal linker and a C-terminal linker and the second domain comprising a N-terminal linker and a C-terminal linker are a first domain and a second domain as defined herein above.

[0418] In some embodiments, the heavy chain variable region VH and the light chain variable region VL constitute an antigen binding site that specifically binds to an antigen. In some embodiments, the heavy chain variable region VH and the light chain variable region VL are a VH and a VL as defined herein above. In some embodiments, the heavy chain variable region VH1 and the light chain variable region VL1 constitute a first antigen binding site that specifically binds to a first antigen, and the heavy chain variable region VH2 and the light chain variable region VL2 constitute a second antigen binding site that specifically binds to a second antigen.

[0419] In some embodiments, the first antigen is HER3 and the second antigen is EGFR. In some embodiments, the first antigen is EGFR and the second antigen is HER3. In some embodiments, the first antigen is HER3 and the second antigen is C-met. In some embodiments, the first antigen is C-met and the second antigen is HER3. In some embodiments, the first antigen is HER3 and the second antigen is PDL1.

[0420] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3 and a second antigen EGFR, wherein VH1 and VL1 are a VH and a VL, respectively, as defined in "Antigen binding site that specifically binds to HER3" in section III "Antigens and antigen binding sites" herein, VH2 and VL2 are a VH and a VL, respectively, as defined in "Antigen binding site that specifically binds to EGFR" in section III "Antigens and antigen binding sites" herein; and the first domain and the second domain are a combination of a first domain and a second domain as defined in section I "Combination of a first domain and a second domain" herein, e.g. a combination of Table A, Table B, Table C and Table D;

[0421] For example, the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein

[0422] (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain;

[0423] (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus;

[0424] (iii) the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; or

[0425] (iv) the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9; or

[0426] for example, the first domain is a SIRPa chain and the second domain is a CD47 chain, wherein

[0427] (i) the SIRPa chain comprises the mutation S98C compared to the parent SIRPa chain and the CD47 chain comprises the mutation combination N5K, N16D, N32S, D46C, N55Q, N93K compared to the parent CD47 chain;

[0428] (ii) the SIRPa chain comprises the mutation S98C compared to the parent SIRPa chain and comprises an N-terminal linker G4S at the N-terminus and a C-terminal linker GE at the C-terminus and the CD47 chain comprises the mutation combination N5K, N16D, N32S, D46C, N55Q, N93K compared to the parent CD47 chain and comprises an N-terminal linker G4S at the N-terminus and a C-terminal linker GE at the C-terminus

[0429] (iii) the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44.

[0430] (iv) the SIRPa chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, and the CD47 chain comprises an amino acid sequence as set forth in SEQ ID NO: 50.

[0431] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen EGFR, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0432] the first heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 135,

[0433] the first light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 139;

[0434] the second heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 131; and / or

[0435] the second light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 73.

[0436] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen EGFR, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0437] the first heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 135,

[0438] the first light chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 139;

[0439] the second heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 131; and

[0440] the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 131, and / or

[0441] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen EGFR, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0442] the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 136,

[0443] the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 141;

[0444] the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 131, and / or

[0445] the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 73.

[0446] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen EGFR, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0447] the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 136,

[0448] the first light chain comprises the amino acid sequence set forth in SEQ ID NO: 141;

[0449] the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 131, and

[0450] the second light chain comprises the amino acid sequence set forth in SEQ ID NO: 73.

[0451] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen EGFR and to a second antigen HER3, wherein VH1 and VL1 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind to EGFR" in section III "Antigens and antigen binding sites" herein, VH2 and VL2 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind to HER3" in section III "Antigens and antigen binding sites" herein, and

[0452] The first domain and the second domain are a combination of a first domain and a second domain as defined in section I "Combination of a first domain and a second domain" herein, e.g. a combination of Table A, Table B, Table C and Table D;

[0453] For example, the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein

[0454] (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain;

[0455] (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus;

[0456] (iii) the PDL2 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 3; or

[0457] (iv) the PDL2 chain comprises the amino acid sequence as shown in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence as shown in SEQ ID NO: 9.

[0458] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen EGFR and to a second antigen HER3, and it comprises or consists of a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein

[0459] the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 134,

[0460] the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 140;

[0461] the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 132; and / or

[0462] the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 75.

[0463] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen EGFR, and a second antigen HER3, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0464] the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 134,

[0465] the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 140;

[0466] the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132; and

[0467] the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0468] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen C-met and a second antigen HER3, wherein VH1 and VL1 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind to C-met" in section III "Antigens and antigen binding sites" herein, VH2 and VL2 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind to HER3" in section III "Antigens and antigen binding sites" herein, and

[0469] The first domain and the second domain are a combination of a first domain and a second domain as defined in section I "Combination of a first domain and a second domain" herein, e.g. a combination of Table A, Table B, Table C and Table D;

[0470] For example, the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein

[0471] (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain;

[0472] (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus;

[0473] (iii) the PDL2 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 3; or

[0474] (iv) the PDL2 chain comprises the amino acid sequence as shown in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence as shown in SEQ ID NO: 9.

[0475] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen C-met, and a second antigen HER3, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0476] the first heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 138,

[0477] the first light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 96;

[0478] the second heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 132; and / or

[0479] the second light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 75.

[0480] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen C-met, and a second antigen HER3, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0481] the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 138,

[0482] the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96;

[0483] the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132; and

[0484] the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75.

[0485] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3 and a second antigen C-met, wherein VH1 and VL1 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind HER3" in section III of the present text, VH2 and VL2 are a VH and VL, respectively, as defined in "Antigen binding sites that specifically bind C-met" in section III of the present text, and

[0486] The first domain and the second domain are a combination of a first domain and a second domain as defined in section I of the present text "Combination of a first domain and a second domain", e.g. a combination of Table A, Table B, Table C and Table D;

[0487] For example, the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein

[0488] (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain;

[0489] (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parent PDL2 chain and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parent PD1 chain and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus;

[0490] (iii) the PDL2 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 3; or

[0491] (iv) the PDL2 chain comprises the amino acid sequence as shown in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence as shown in SEQ ID NO: 9.

[0492] In some embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3 and a second antigen C-met, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein

[0493] the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 135,

[0494] the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 139;

[0495] the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 133; and / or

[0496] the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 71.

[0497] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen C-met, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0498] the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135,

[0499] the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139;

[0500] the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 133; and

[0501] the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71.

[0502] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3 and a second antigen PDL1, wherein VH1 and VL1 are a VH and a VL, respectively, as defined in "Antigen binding sites that specifically bind to HER3" in section III "Antigens and antigen binding sites" herein, VH2 and VL2 are a VH and a VL, respectively, as defined in "Antigen binding sites that specifically bind to PDL1" in section III "Antigens and antigen binding sites" herein, and

[0503] The first domain and the second domain are a combination of a first domain and a second domain as defined in section I "Combination of a first domain and a second domain" herein, e.g. a combination of Table A, Table B, Table C and Table D;

[0504] For example, the first domain is a CD47 chain and the second domain is a SIRPa chain, wherein

[0505] (i) the CD47 chain comprises the combination of mutations N5K, N16D, E29C, N32S, N55Q, N93E compared to the parental CD47 chain and the SIRPa chain comprises the mutation R69C compared to the parental SIRPa chain;

[0506] (ii) the CD47 chain comprises the combination of mutations N5K, N16D, E29C, N32S, N55Q, N93E compared to the parental CD47 chain and comprises an N-terminal linker G4S at the N-terminus and an C-terminal linker AKEPKSA (Seq ID NO: 67) at the C-terminus; and the SIRPa chain comprises the mutation R69C compared to the parental SIRPa chain and comprises an N-terminal linker G4S at the N-terminus and an C-terminal linker EPKSA (Seq ID NO: 68) at the C-terminus;

[0507] (iii) the CD47 chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 35 and the SIRPa chain comprises or consists of the amino acid sequence as shown in SEQ ID NO: 59; or

[0508] (iv) the CD47 chain comprises the amino acid sequence as shown in SEQ ID NO: 47 and the SIRPa chain comprises the amino acid sequence as shown in SEQ ID NO: 65.

[0509] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3 and a second antigen PDL1, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein

[0510] the first heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 172,

[0511] the first light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 173;

[0512] the second heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 170; and / or

[0513] the second light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 171.

[0514] In some specific embodiments, the bispecific antibody of the present disclosure specifically binds to a first antigen HER3, and a second antigen EGFR, and comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein

[0515] the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 172,

[0516] the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 173;

[0517] the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 170; and

[0518] the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 171.

[0519] The polypeptide complex, e.g., multispecific antibody such as bispecific antibody, of the present disclosure has reduced light and heavy chain mispairing compared to an antibody that does not have first and second domain heterodimerization. The polypeptide complex, e.g., multispecific antibody such as bispecific antibody, of the present disclosure retains the activity, e.g., affinity and / or endocytosis activity, of the original antibody compared to an antibody that does not have first and second domain heterodimerization.

[0520] II. Polypeptide complex nucleic acids, vectors and host cells, and methods of making

[0521] In another aspect of the disclosure, the disclosure provides methods of making a polypeptide complex, for example comprising:

[0522] a) introducing nucleic acids encoding each chain of the polypeptide complex into a host cell;

[0523] b) expressing and assembling the polypeptide complex within the host cell;

[0524] Optionally, the polypeptide complex is purified, for example by affinity chromatography such as Protein A purification.

[0525] The disclosure also provides nucleic acids encoding a polypeptide complex of the disclosure, or any one chain or any monomer or domain thereof. The nucleic acids of the disclosure can also comprise a segment encoding a secretion signal peptide, thereby directing secretion expression of the polypeptide complex or each chain thereof.

[0526] The disclosure also provides vectors comprising the nucleic acids of the disclosure. In one embodiment, the vector is an expression vector, for example a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the disclosure is a pCNDA vector, for example a pCNDA3.0 expression vector.

[0527] The disclosure also provides host cells comprising the nucleic acids or the vectors. Host cells suitable for replication and support of expression of polypeptide complexes of the disclosure, such as multispecific antibodies, for example bispecific antibodies, are well known in the art. Such cells can be transfected or transduced with a particular expression vector, and large quantities of vector-containing cells can be grown for seeding large scale fermentors to obtain sufficient quantities of polypeptide complex for clinical applications. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell or a mammalian cell (for example a CHO cell or a 293 cell, for example a CHOK1 cell or an Expi293 cell). In some embodiments, the host cell is prokaryotic, for example an E. coli cell.

[0528] III. Immunoconjugates and pharmaceutical compositions and uses

[0529] The disclosure also relates to immunoconjugates comprising a polypeptide complex of the disclosure and one or more antitumor compounds, for example a cytotoxic agent or a chemotherapeutic agent or a toxin or a label (for example a detectable label). In some embodiments, when the polypeptide complex is an antibody, for example a monospecific antibody or a bispecific antibody, the immunoconjugate can be an antibody drug conjugate, for example an ADC.

[0530] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide complex or immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient. It is a further object of the present disclosure to provide a method of manufacturing a pharmaceutical composition of the present disclosure, said method comprising combining a polypeptide complex or immunoconjugate as described herein, or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers.

[0531] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present disclosure are those suitable for use in contact with the tissues of human beings without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0532] The pharmaceutical composition can be administered in any form that will allow it to achieve the desired prevention, alleviation or cure of the symptoms in a human or animal patient. For example, various suitable dosage forms can be prepared depending on the route of administration.

[0533] In other embodiments, the administration of the polypeptide complex or immunoconjugate or pharmaceutical composition of the present disclosure can be combined with one or more additional therapeutic methods. Said additional therapeutic methods can be selected from, but are not limited to, radiotherapy, chemotherapy, immunotherapy, or a combination thereof. In other embodiments, the polypeptide complex or immunoconjugate or pharmaceutical composition of the present disclosure can be administered in combination with one or more other therapeutic agents.

[0534] In some embodiments, when the polypeptide complex or immunoconjugate or pharmaceutical composition of the present disclosure is used to treat a tumor, said other therapeutic agents are selected from chemotherapeutic agents, cytotoxic agents, small molecule drugs, angiogenesis inhibitors, immunomodulatory agents or other anti-tumor antibodies.

[0535] The present disclosure also relates to a pharmaceutical preparation comprising a polypeptide complex or immunoconjugate of the present disclosure, or a pharmaceutically acceptable form thereof, or a mixture thereof, as active ingredient, or a pharmaceutical composition of the present disclosure.

[0536] The present disclosure also relates to a pharmaceutical combination or combination product comprising a polypeptide complex or immunoconjugate or pharmaceutical composition of the present disclosure, and one or more other therapeutic agents. In some embodiments, when the polypeptide complex or pharmaceutical composition of the present disclosure is used to treat a tumor, said other therapeutic agents are selected from chemotherapeutic agents, cytotoxic agents, small molecule drugs, angiogenesis inhibitors, immunomodulatory agents or other anti-tumor antibodies.

[0537] The present disclosure also relates to a kit-of-parts comprising a pharmaceutical combination or combination product of the present disclosure, preferably said kit is in the form of a pharmaceutical dosage unit. Thereby dosage units can be provided according to a dosing regimen or pharmaceutical administration interval.

[0538] In one embodiment, the kit-of-parts of the present disclosure comprises in the same package:

[0539] - a first container containing a pharmaceutical composition comprising a polypeptide complex or immunoconjugate or a pharmaceutical composition of the present disclosure;

[0540] - a second container containing a pharmaceutical composition comprising a further therapeutic agent.

[0541] In some embodiments, when the polypeptide complex or immunoconjugate or pharmaceutical composition of the present disclosure is used for the treatment of a tumor, the further therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a small molecule drug, an angiogenesis inhibitor, an immunomodulatory agent or another anti-tumor antibody.

[0542] In yet another aspect, the present disclosure provides the use of a polypeptide complex or immunoconjugate as described herein or a pharmaceutical composition as described herein for the manufacture of a medicament for the treatment and / or prevention of a disease or disorder. In yet another aspect, the present disclosure provides a method of treating and / or preventing a disease or disorder, comprising administering to a subject in need thereof a polypeptide complex or immunoconjugate as described herein or a pharmaceutical composition as described herein. In yet another aspect, the present disclosure provides a polypeptide complex or immunoconjugate as described herein or a pharmaceutical composition for use in therapy, e.g. for use in the treatment or prevention of a disease or disorder.

[0543] In some embodiments, the disease and / or disorder can be selected from the group of proliferative diseases and / or conditions, metabolic diseases and / or conditions, inflammatory diseases and / or conditions and neurodegenerative diseases and / or conditions.

[0544] In some embodiments, the disease and / or disorder is a tumor, e.g. a cancer. In some embodiments, the disease and / or disorder is an autoimmune disease and / or disorder. In some embodiments, the disease and / or disorder is selected from an inflammatory disease and / or disorder.

[0545] In some embodiments, the tumor is a solid tumor or a hematological tumor.

[0546] In some embodiments, the tumor or cancer expresses or overexpresses one or more antigens targeted by the polypeptide complex of the present disclosure, e.g. a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the tumor-associated antigen is an immune checkpoint molecule. In some embodiments, the antigen is an antigen as described in the present disclosure. In some embodiments, the tumor-associated antigen is selected from C-met, EGFR, PDL1 or HER3.

[0547] In some embodiments, the tumor or cancer is a malignant tumor with abnormal activation of the Met pathway, for example, a solid tumor, for example, brain cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, liver cancer, skin cancer, prostate cancer, or soft tissue cancer, etc.

[0548] In some embodiments, the tumor or cancer is a malignant tumor with EGFR expression or overexpression in tumor cells, for example, glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, or breast cancer, etc.

[0549] In some embodiments, the tumor or cancer is a malignant tumor with HER3 expression in tumor cells, for example, breast cancer, ovarian cancer, colon cancer, gastric cancer, lung cancer, skin cancer, or pancreatic cancer, etc.

[0550] In some embodiments, the tumor or cancer is selected from brain cancer, breast cancer, colorectal cancer, colon cancer, gastric cancer, head and neck cancer, lung cancer, liver cancer, skin cancer, prostate cancer, soft tissue cancer, glioma, renal cancer, pancreatic cancer, ovarian cancer, or skin cancer.

[0551] In yet another aspect, the disclosure provides a method for detecting a first antigen and / or a second antigen using a polypeptide complex as described herein. In certain embodiments, the method comprises detecting the presence of the antigen in a biological sample. In certain embodiments, the method comprises contacting a biological sample with a polypeptide complex as described herein under conditions in which it binds thereto, and detecting whether a complex is formed between the polypeptide complex and the antigen. Formation of a complex indicates the presence of the antigen. The method can be an in vitro or in vivo method. In one embodiment, the polypeptide complex is used to select a subject suitable for treatment with the polypeptide complex, for example, where the antigen is a biomarker used to select the subject. In some embodiments, a labeled polypeptide complex is provided. Labels include, but are not limited to, labels or moieties that are directly detected, such as fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as moieties that are indirectly detected, such as enzymes or ligands, for example, by enzymatic reaction or molecular interaction. In some embodiments provided herein, the sample is obtained prior to treatment with a polypeptide complex or immunoconjugate, pharmaceutical composition, pharmaceutical combination, or kit of the disclosure. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies. In some embodiments, the antigen bound by the polypeptide complex is detected prior to treatment, for example, prior to initiation of treatment or prior to a certain treatment after a treatment interval.

[0552] IV. Definitions

[0553] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0554] To facilitate a better understanding of the present disclosure, certain key terms are specifically defined as follows. As used herein, and unless otherwise expressly specified, all terms used herein have the meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. With respect to the definitions and terms of art, the skilled artisan can refer to Current Protocols in Molecular Biology (Ausubel) at least in part.

[0555] Abbreviations for amino acid residues follow the standard 3-letter and / or 1-letter codes used in the art to designate one of the 20 common L-amino acids.

[0556] As used herein, including the claims, the singular form "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0557] The term "about" when used in connection with a numerical value means a range encompassing numerical values less than 5% of the lower limit of the specified numerical value and greater than 5% of the upper limit of the specified numerical value, including but not limited to ± 5%, ± 2%, ± 1%, and ± 0.1%.

[0558] The term "and / or" should be understood to mean either one of the items or any combination of the items in the list.

[0559] As used herein, the term "or" is to be interpreted as having the same meaning as "and / or" as defined above. For example, when used in the phrases "A or B" or "A and / or B" the term "or" should be interpreted as meaning either one of A or B, or any combination of A and B.

[0560] As used herein, the term "comprising" or "including," or "having" means including, but not limited to, the stated elements, integers or steps, and that one or more of the stated elements, integers or steps can be present, but not exclude the presence of additional elements, integers or steps. In this document, the terms "comprising" and "including" when used in the specification are also to be construed to cover the case where the stated elements, integers or steps are comprised or included, but not to exclude the presence of additional elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to cover an antibody variable region consisting of the particular sequence.

[0561] In the present application, the term "antibody" generally refers to an immunoglobulin that is reactive to a specified protein or peptide or fragment thereof. The antibody can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibody. The antibody can have a heavy chain constant region selected from, for example, IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (K) or lambda (l). The antibodies of the present application can be derived from any species. The term "antibody" can include intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule that exhibits the desired biological activity.

[0562] In the present application, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy or light chain that is involved in binding the antibody to the antigen. In the present application, the term "variable" generally refers to the fact that certain portions of the variable domain sequences are highly divergent between antibodies, contributing to the binding and specificity of each particular antibody to its particular antigen. Variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) of the light chain variable domain and three segments called CDRs or HVRs of the heavy chain variable domain, referred to as LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively. The more highly conserved portions of the variable domains are referred to as framework regions (FRs). The variable domains of the heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a beta-sheet configuration, connected by three CDR loops. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of antibodies.

[0563] In the art, the variable regions of antibodies or the CDRs of antibodies can be encoded or delineated by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, Bethesda, MD (1991)), the Chothia numbering scheme and definition rules based on the location of structural loop regions (see, Al-Lazikani et al., J Mol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules based on alignment of amino acid sequences of germline V genes by efranc et al., and Honnegers numbering scheme (AHo’s), Martin numbering scheme, Gelfand numbering scheme, and the like, see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018.

[0564] The term“full-length antibody” refers to an immunoglobulin molecule comprising at least four peptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as naturally occurs. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH or VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0565] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present (the numbering in this paragraph is according to the EU numbering system, also referred to as EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0566] As used herein, amino acid positions of the entire variable region of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as "Kabat numbering".

[0567] As used herein, when referring to amino acid positions in domains of an antibody other than the variable region (e.g., constant regions, e.g., Fc region), numbering is according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to herein as "EU numbering". When position numbering and / or amino acid residues are assigned to a particular antibody isotype, it is intended that the corresponding position and / or amino acid residue of any other antibody isotype be determined by one of ordinary skill in the art.

[0568] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "substituted" amino acid residue. The substituted residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (He); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Also encompassed in the definition of amino acid substitution herein is the replacement with one or more non-naturally occurring amino acid residues. "Non-naturally occurring amino acid residues" refer to residues other than those listed above that are capable of covalently bonding to an adjacent amino acid residue in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0569] The polypeptide complexes of the present disclosure can comprise a linker. The term "linker" or "spacer" or "linking domain" as used herein can be used interchangeably herein to refer to the boundary or boundary region of the fusion or combination of two polypeptide sequences. Examples of linkers that establish covalent linkage between different portions of a multispecific antibody include peptide linkers and non-protein polymers. For example, a linker can comprise at least a portion of a C-terminal fragment from a first fusion polypeptide fused to at least a portion of an N-terminal fragment from a second fusion polypeptide, with or without additional spacers therebetween. For example, a linker can be rich in glycine and proline residues. For example, a spacer having a single or repeating sequence of threonine / serine and glycine, GGGGS, TGGGG, SGGGG, or tandem repeats thereof (e.g., 2, 3, 4, 5 or more repeats). In certain embodiments, the spacer comprises GGGGS or a 2- or 3- or more tandem repeats thereof. In some embodiments herein, when referring to a C-terminal linker, it can also be located at the C-terminus of only one polypeptide as a boundary, without linking to another polypeptide.

[0570] The term "immunoconjugate" or "drug conjugate" refers to a novel drug in which a ligand is linked to a biologically active drug through a stable linking unit. In some embodiments, the immunoconjugate is an antibody drug conjugate. An "antibody drug conjugate" (ADC) as described herein is an antibody or antibody fragment linked to a biologically active drug through a linking unit.

[0571] In the present context, "heterodimeric Fc scaffold" refers to a scaffold comprising or formed upon dimerization of two different Fc regions, which can have a domain binding an antigen (e.g. a heavy chain variable region and / or a light chain variable region of an antibody or an antigen binding fragment of an antibody that can bind to an antigen) attached at its N- or C-terminus for the construction of a polypeptide complex of the disclosure, e.g. a multispecific antibody or a bispecific antibody.

[0572] Amino acid mutations are denoted using (original amino acid, amino acid position, mutated amino acid). For example, "53C" or "K53C" means that the amino acid at the position corresponding to parent position 53 is substituted with cysteine (C). When multiple possibilities exist for a mutation at a particular position, this is denoted herein by the symbol " / ". For example, the mutation "44T / C" means that the amino acid residue at the position corresponding to parent position 44 can be replaced with a T or a C residue.

[0573] It should be noted that when describing a mutation, the original amino acid at a particular position can be the one described, or it can be another amino acid at the corresponding position. For example, when a mutation is described using "K53C", it has the same meaning as "53C", i.e. the amino acid at the position corresponding to 53 in the parent is mutated to cysteine, regardless of the original amino acid being at the position 53 of the variable region, and not necessarily being lysine K.

[0574] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0575] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in the candidate sequence that have the same residue as the reference sequence across the entire length of the aligned sequences, after aligning the sequences (introducing gaps, if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment for purposes of determining percent amino acid sequence identity can be achieved using various methods, for example, using publically available computer software such as BLAST, BLAST-2, ALIGN, or the MEGALIGN DNA STAR software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0576] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to refer to a polymer of amino acid residues, or a collection of polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. A polypeptide sequence is depicted typically with its left-hand end as the amino terminus (N terminus, N-terminus) and its right-hand end as the carboxy terminus (C terminus, C-terminus).

[0577] A "polypeptide complex" as described herein refers to a complex comprising one or more polypeptides associated with performing a certain function. In some embodiments, the polypeptide complex is or comprises an antibody or antigen-binding fragment thereof that is capable of specifically binding to an antigen. A polypeptide complex comprises one or more, preferably two or more polypeptide chains. In some embodiments, the polypeptide complex is a monospecific antibody. In some embodiments, the polypeptide complex is a multispecific antibody, e.g., a bispecific antibody.

[0578] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more target binding sites, each of which binds to the same site or structure of the same target or to the same epitope of the same antigen.

[0579] As used herein, the term "multispecific" antibody refers to an antibody that has at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0580] The term "binding region" or "binding site" or "binding domain" with respect to an antigen refers to the region or portion of a specific protein molecule (e.g., an antibody molecule) that is capable of specific binding to an antigen, an antigen binding site can be a ligand binding domain portion that is capable of directly binding to an antigen, or a domain comprising an antibody variable region that is capable of directly binding to an antigen. In one embodiment, an antigen binding site of the present disclosure comprises or consists of a heavy chain variable region and a light chain variable region.

[0581] When reference is made herein to an "antigen binding portion" in a polypeptide complex, it refers to the portion of the polypeptide complex that binds to an antigen. An antigen binding portion comprises an antigen binding site, or other domain, e.g., an Fc region. A polypeptide complex can comprise one, two, or more antigen binding portions.

[0582] In the context of this document, different antigen binding moieties or antigen binding sites also encompass binding moieties that bind to different epitopes of the same protein. Thus, when referring to a first antigen binding moiety and a second antigen binding moiety in a polypeptide complex, this can either mean a first antigen binding moiety and a second binding moiety that specifically bind to different antigens, or a first epitope binding moiety and a second epitope binding moiety that bind to different epitopes of the same antigen, wherein the first and second epitopes can be from the same antigen (i.e. different epitopes on the same antigen) or different antigens (i.e. epitopes on different antigens).

[0583] When referring to a "first antigen binding moiety" or a "first antigen binding site" in a polypeptide complex, this refers to a binding moiety or binding site that binds to a first antigen, without intending to limit the number of such antigen binding moieties or binding sites contained in an antibody, e.g. a multispecific antibody or a bispecific antibody can comprise one or more than one first antigen binding moiety or first antigen binding site. For example, a bispecific antibody comprises a first antigen binding moiety and a second antigen binding moiety, but can comprise one or more than one first antigen binding moiety and one or more than one second antigen binding moiety.

[0584] The term "operably linked" means that two or more biological sequences are linked in a manner that allows them to function in their intended manner, with or without a spacer region (also known as a linker, linker sequence). When used in reference to a polypeptide, the term indicates that the polypeptide sequences are linked in a manner that allows the product to have the intended biological function of the linked sequences, with or without a spacer region between the two sequences. For example, an antibody variable region can be operably linked to a constant region to form a stable product with antigen binding activity. The term can also be used in reference to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g. a promoter, enhancer, silencer sequence, etc.), the term indicates that the polynucleotide sequences are linked in a manner that allows the polypeptide to be expressed from the polynucleotide under the control of the regulatory sequence.

[0585] The term "dimerization domain" refers to a polypeptide domain that is capable of promoting association with one another to form a dimer. In some embodiments, a first dimerization domain can associate with a second dimerization domain. The association can be by any suitable means of binding or linkage or bonding; for example, by a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or combinations thereof. Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody CH3 domains, antibody Fc regions, and other suitable protein monomers or polypeptides that are capable of dimerizing and interacting with one another.

[0586] The term "interaction domain" refers to a domain of a polypeptide that is capable of facilitating the interaction or association of two or more homologous or heterologous polypeptides. For example, an interaction domain is a dimerization domain that facilitates association with one another to form a dimer. A domain of an inter-protein interaction is a domain of a polypeptide that is capable of interacting or associating with two or more proteins, for example, the IgV-like domain in a PD1 protein or a fragment thereof, is capable of forming a complex with the IgV-like domain in a PDL1 or PDL2 protein or a fragment thereof through intermolecular interactions. Alternatively, the IgV-like domain in a CD47 protein or a fragment thereof, is capable of forming a complex with the IgV-like domain in a SIRPa protein or a fragment thereof through intermolecular interactions.

[0587] The term "pharmaceutical composition" generally refers to a composition comprising one or more polypeptide complexes described herein or immunoconjugates thereof, and other components such as pharmaceutically acceptable carriers and excipients. The pharmaceutical composition can be formulated to facilitate administration to an organism, to facilitate absorption of the active ingredients, and to thereby facilitate biological activity. The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0588] The term "pharmaceutically acceptable carrier" generally refers to a carrier or vehicle for administration of a therapeutic agent, such as an antibody or polypeptide, a gene, and other therapeutic agents. The term refers to a carrier that does not itself induce the production of antibodies to the individual receiving the composition. The carrier can be any pharmaceutical carrier that can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactivated viruses. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a pharmaceutical composition can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances can also be present in these carriers such as wetting or emulsifying agents, pH buffering substances, and the like.

[0589] The term "small molecule drug" refers to an organic compound of low molecular weight that is capable of modulating a biological process, particularly altering or preventing a pathological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, and more preferably less than 500 D. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can penetrate cells more readily than macromolecules, be less susceptible to degradation, and be less likely to elicit an immune response.

[0590] The term "treatment" generally refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. Therapeutic benefit includes, but is not limited to, eradication, inhibition, reduction, or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved with the eradication, inhibition, reduction, or amelioration of one or more signs or symptoms associated with the underlying disorder such that an improvement is observed in the patient, but the patient can still be afflicted with the underlying disorder.

[0591] The term "prevention" generally refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more physiological symptoms of a disease, even though the disease has not yet been diagnosed.

[0592] The term "subject" or "patient" or "individual" generally refers to a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or senior adult)) and / or other primate (e.g., a cynomolgus monkey, a rhesus monkey); a mammal, including commercially relevant mammals such as a cow, pig, horse, sheep, goat, cat, and / or dog; and / or a bird, including commercially relevant birds such as a chicken, duck, goose, quail, and / or turkey.

[0593] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to the amount of a polypeptide complex of the disclosure or an immunoconjugate thereof, administered alone or in combination with other therapeutic agents or treatments, to a cell, tissue, or subject that is effective to prevent or ameliorate the symptoms of or the progression of one or more diseases or conditions. A therapeutically effective dose also refers to the amount of an agent that will result in an improvement of symptoms, e.g., an amount that treats, cures, or ameliorates the relevant medical condition or increases the rate of treatment, cure, or amelioration of such a condition. When administered to an individual, a therapeutically effective dose refers only to the administered ingredient. When administered in combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that will result in the therapeutic effect, whether administered in combination, sequentially, or concurrently. An effective amount of a therapeutic agent will result in an improvement of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in a diagnostic standard or parameter.

[0594] It should be understood that the disclosure is not limited to the particular methodology, protocols, and reagents described herein as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. Examples

[0595] Preparation and purity test of the antibodies of Example 1

[0596] Complex designed for replacing CH1 / CL domain

[0597] Based on PD1 / PDL1 complex, PD1 / PDL2 complex, CD47 / SIRPα complex respectively, chains with natural intermolecular interaction were obtained, and then corresponding mutations were designed in these chains to obtain chains as shown in Table 1 below. The specific sequences are shown in the sequence summary table.

[0598] Table 1: PD1 chain, PDL1 chain, PDL2 chain, and SIRPα chain, CD47 chain amino acid mutations

[0599] Preparation of monospecific antibodies

[0600] Using the above-mentioned mutated or structurally modified PD1 chain, PDL1 / PDL2 chain, SIRPα chain, CD47 chain, CH1 / CL replaced antibodies were constructed (structure see Table 2), wherein Cm1-Cm21 are anti-C-met antibodies, Hm1 and Hm2 are anti-Her3 antibodies, and Em1 is an anti-EGFR antibody.

[0601] The full-length coding nucleic acids of the heavy chain and the light chain were introduced into pcDNA3.0 plasmids respectively, and then transfected into TOP10 E. coli cells. The MN plasmid extraction kit was used to obtain the plasmids. The obtained plasmids were transfected into CHOK1 cells using PEI, and the corresponding antibodies were expressed and purified using Protein A.

[0602] The purity of the above-mentioned antibodies was tested using SEC-UPLC method. The specific method parameters are shown in the table below.

[0603] The purity of the CH1 / CL replaced antibodies was tested, and the experimental results are shown in Table 2.

[0604] The results show that after the CH1 / CL is replaced, the antibody still maintains a normally assembled structure.

[0605] Table 2: Antibody purity results after CH1 / CL replacement

[0606] Note: For example, antibody Cm2 in the table means that the heavy chain CH1 of Cm1 antibody (CmH (Seq ID NO: 70), CmL (Seq ID NO: 71)) is replaced by PD1 chain-2 (Seq ID NO: 2), and the light chain CL is replaced by PDL1 chain-2 (Seq ID NO: 15), and the other parts remain the same as Cm1, and the obtained antibody, and the other is the same as the above.

[0607] Preparation of bispecific antibodies

[0608] Further, using the above-mentioned mutations, a plurality of bispecific antibody assembly experiments were carried out, and the modified PD1 chain, PDL1 / PDL2 chain, SIRPα chain, CD47 chain were constructed to obtain CH1 / CL replaced bispecific antibodies. As described above, the preparation of bispecific antibodies similar to monoclonal antibodies is shown in Table 3 below, and the respective domains of the heavy chain and light chain are shown in Table 3 below, and the sequences of each chain are shown in the sequence listing and SEQ ID NO.

[0609] Similarly, the above-mentioned bispecific antibodies were detected for purity using the SEC-UPLC method, and the experimental results are shown in Table 3. The results show that after the CH1 / CL is replaced, the antibody still maintains a normally assembled structure.

[0610] Table 3: Purity results of bispecific antibodies after CH1 / CL replacement

[0611] Note: Em1H and Em1L are the heavy chain and light chain of Em1, respectively, Hm1H and Hm1L are the heavy chain and light chain of Hm1, respectively, Cm1H and Cm1L are the heavy chain and light chain of Cm1, respectively, and PmH and PmL are the heavy chain and light chain of the PDL1 antibody, respectively, and the light chain variable region and heavy chain variable region thereof are from WO2021036929A1.

[0612] In addition, the heavy chain of the control antibody BsAb-9 (DBXT005-01 in PCT / CN2024 / 106435) is the C-terminal coupling of (G4S)*3Scfv constructed by the heavy chain variable region and the light chain variable region of the Hm1 antibody, the sequence of the heavy chain 1 is Seq ID NO: 137, and the sequence of the heavy chain 2 is SEQ ID NO: 142; the light chain is Em1, and the sequence is Seq ID NO: 73. It is detected that the purity of BsAb-9 is 89.5%.

[0613] Example 2, functional verification of monospecific antibodies and bispecific antibodies

[0614] The molecules prepared in Example 1 were selected for functional verification.

[0615] 2.1 Test of antibody affinity to cells

[0616] First, flow cytometry was applied to detect the affinity of antibodies to cells, and the specific experimental steps are as follows:

[0617] The target cells NCI-H1975 (R&D Systems RD00700) were collected and resuspended in FACS experiment buffer (99% DPBS + 1% fetal bovine serum).

[0618] The target cell density was adjusted with FACS experiment buffer (100,000 cells / well, 50 μl / well), and the cell suspension was transferred to a 96-well experiment plate.

[0619] The test and control samples were prepared with FACS experiment buffer (10 nM and 1 nM), wherein

[0620] Test sample: the above-mentioned antibody;

[0621] Control sample: human IgG1 (R&D Systems ISO antibody);

[0622] Detection antibody: mouse antibody, targeting human antibody Fc fragment

[0623] Only cells: only cells without working solution.

[0624] Transfer the test / control sample working solution to the corresponding wells of the 96-well experiment plate (50 μl / well), and incubate the experiment plate at 4°C for 1 hour.

[0625] Remove the experiment plate, centrifuge, and discard the supernatant.

[0626] Wash the cells with FACS experiment buffer.

[0627] Transfer the secondary antibody working solution (rabbit anti-mouse antibody, targeting mouse anti-Fc fragment) to the corresponding wells of the 96-well experiment plate.

[0628] Incubate the experiment plate at 4°C for 20 minutes, avoiding light.

[0629] Remove the experiment plate, centrifuge, and discard the supernatant. Wash the cells with FACS experiment buffer.

[0630] Use a BD flow cytometer for detection.

[0631] The results are shown in Figures 1-3. From the results, it can be seen that all CH / CL1 replaced single and double antibodies are equivalent to the corresponding single antibody or traditional form (fab+scfv) double antibody in terms of binding to the corresponding antigen of the target cells.

[0632] 2.2 Antibody internalization experiment test

[0633] Antibody internalization experiment steps (Incucyte method)

[0634] Digest, collect target cells NCI-H1975 and resuspend target cells with experimental buffer (corresponding complete medium: 90% RPMI-1640 Medium + 10% FBS).

[0635] Adjust target cell density and transfer cell suspension into 96-well experimental plate (5E3 cells / well).

[0636] Incubate experimental plate in cell incubator (37°C / 5% CO2) overnight.

[0637] Prepare test / control working solution and labeling reagent working solution (4x, source Incucyte Human Fabfluor-pH Red Antibody Labeling Reagent, Sartorius, Cat# 4722) with experimental buffer, wherein test: above-mentioned antibody;

[0638] Control: Human IgG1 (Vivo Biosciences ISO antibody).

[0639] Mix test / control working solution and labeling reagent working solution at 1:3 molar ratio, 1:1 volume ratio, and incubate in cell incubator (37°C / 5% CO2) for 15 minutes to allow full conjugation.

[0640] Transfer conjugated mixture working solution into corresponding wells of 96-well experimental plate.

[0641] Incubate experimental plate in cell incubator (37°C / 5% CO2) for a certain period of time, and take pictures at designated time points using Incucyte instrument that meets the detection wavelength band.

[0642] Antibody internalization experiment data analysis

[0643] Experimental raw data and results are analyzed and exported via Live-Cell Analysis System. Raw data are further analyzed and plotted using Microsoft Office Excel 2016 and GraphPad Prism 6 software. In GraphPad Prism 6 software, the following four-parameter equation can be used to obtain the relative numerical value of EC50: Y = Bottom + (Top-Bottom) / (1 + 10^((LogEC50-X) x HillSlope)). Wherein, X = sample concentration, Y = Total Red Object Integrated Intensity (RCU x pm2) (Figure). 2

[0644] ​Results are shown in Figures 4-6 and Tables 4-6. All CH / CLl -substituted mono, di- Abs were comparable to the corresponding mAbs or traditional (fab+scfv) diabodies in mediating endocytosis.

[0645] Table 4

[0646] Table 5

[0647] Table 6

[0648] Sequence Summary

Claims

1. A polypeptide complex comprising a first antigen binding moiety comprising a first polypeptide comprising from N-terminus to C-terminus a first heavy chain variable region (VH1), said VH1 operably linked to a first domain (Domain 1); a second polypeptide comprising from N-terminus to C-terminus a first light chain variable region (VL1), said VL1 operably linked to a second domain (Domain 2), wherein said first antigen binding moiety specifically binds to a first antigen, wherein the first domain and the second domain are capable of interacting to form a dimer; wherein (i). the first domain comprises a PD1 chain and the second domain comprises a domain capable of interacting with the PD1 chain, such as a PDL1 chain or a PDL2 chain; (ii). the first domain comprises a domain capable of interacting with the PD1 chain, such as a PDL1 chain or a PDL2 chain, and the second domain comprises a PD1 chain; (iii). the first domain comprises a PDL1 or PDL2 chain and the second domain comprises a domain capable of interacting with the PDL1 or PDL2 chain, such as a PD1 chain; (iv). the first domain comprises a domain capable of interacting with the PDL1 or PDL2 chain, such as a PD1 chain, and the second domain comprises a PDL1 or PDL2 chain; (v). the first domain comprises a CD47 chain and the second domain comprises a domain capable of interacting with the CD47 chain, such as a SIRPa chain; (vi). the first domain comprises a domain capable of interacting with the CD47 chain, such as a SIRPa chain, and the second domain comprises a CD47 chain; (vii). the first domain comprises a SIRPa chain and the second domain comprises a domain capable of interacting with the SIRPa chain, such as a CD47 chain; or (viii). the first domain comprises a domain capable of interacting with the SIRPa chain, such as a CD47 chain, and the second domain comprises a SIRPa chain; wherein the PD1 chain is an extracellular immunoglobulin variable region IgV-like domain of a PD1 protein or a fragment thereof or a variant thereof, the PDL1 chain is an extracellular immunoglobulin variable region IgV-like domain of a PDL1 protein or a fragment thereof or a variant thereof, the PDL2 chain is an extracellular immunoglobulin variable region IgV-like domain of a PDL2 protein or a fragment thereof or a variant thereof, the CD47 chain is an extracellular immunoglobulin variable region IgV-like domain of a CD47 protein or a fragment thereof or a variant thereof, or the SIRPa chain is an extracellular immunoglobulin variable region IgV-like domain of a SIRPa protein or a fragment thereof or a variant thereof.

2. The polypeptide complex of claim 1, wherein the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, or is a modified PD1 chain having a cysteine mutation (i.e. to cysteine) to create an interchain disulfide bond relative to the parent PD1 chain set forth in SEQ ID NO: 1, and / or an N-glycosylation site mutation (i.e. to a non-glycosylated amino acid such as S or D), preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or the binding of the PD1 chain to the PDL1 chain or the PDL2 chain; or the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, or is a modified PDL1 chain having a cysteine mutation (i.e. to cysteine) to create an interchain disulfide bond relative to the parent PDL1 chain set forth in SEQ ID NO: 14, and / or an N-glycosylation site mutation (i.e. to a non-glycosylated amino acid such as T), preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or the binding of the PDL1 chain to the PD1 chain; or the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23, or is a modified PDL2 chain having a cysteine mutation (i.e. to cysteine) to create an interchain disulfide bond relative to the parent PDL2 chain set forth in SEQ ID NO: 23, and / or an N-glycosylation site mutation (i.e. to a non-glycosylated amino acid such as D), preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or the binding of the PDL2 chain to the PD1 chain.

3. The polypeptide complex of claim 1 or 2, wherein the modified PD1 chain has one or more amino acid residue mutations selected from 15S, 24S, 30C, 40S, 44C / T, 59S or 82D or has a mutation combination of N15S, N24S, N40S, C59S, N82D and optionally one or more other mutations, such as 30C and / or 44T / C, wherein the modified PD1 chain mutation site is the ordinal number site relative to the sequence of SEQ ID NO: 1; Preferably, the modified PD1 chain has a mutation combination as follows: N15S, N24S, N40S, K44C, C59S, N82D; N15S, N24S, V30C, N40S, C59S, N82D; N15S, N24S, N40S, K44T, C59S, N82D; or N15S, N24S, N40S, C59S, N82D; More preferably, the modified PD1 chain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-11; or; the modified PDL1 chain has one or more amino acid residue mutations selected from 4A, 17T, 23A, 73A and 104C or has the amino acid residue mutations 17T and optionally one or more further amino acid mutations, e.g. 104C, 4A, 23A or 73A, wherein the modified PDL1 chain mutation sites are sequentially numbered sites relative to sequence SEQ ID NO: 14; Preferably, the modified PDL1 chain has the following combination of mutations: N17T, D104C; or N17T, T4A, K23A, Q73A; more preferably, the modified PDL1 chain comprises or consists of the amino acid sequence recited in SEQ ID NO: 19 or 20; or the modified PDL2 chain has one or more amino acid residue mutations selected from 17D, 44D or 88C or has the amino acid residue mutations 17D and 44D and optionally one or more further amino acid mutations, e.g. 88C, wherein the modified PDL2 chain mutation sites are sequentially numbered sites relative to sequence SEQ ID NO: 23; Preferably, the modified PDL2 chain has the following combination of mutations: N17D, N44D, V88C; or N17D, N44D; more preferably, the modified PDL2 chain comprises or consists of the amino acid sequence recited in any one of SEQ ID NO: 28 or 29.

4. The polypeptide complex of any one of claims 1-3, wherein the PD1 chain, PDL1 chain or PDL2 chain further comprises a linker, e.g. an N-terminal linker at the N-terminus and / or a C-terminal linker at the C-terminus, wherein the N-terminal linker is selected from the N-terminal contiguous fragment of the chain in the protein from which the chain is derived, the N-terminal fragment of the heavy chain constant region CH1 (e.g. when it is N-terminally connected to a heavy chain variable region), the N-terminal fragment of the light chain constant region CL (e.g. when it is N-terminally connected to a light chain variable region), or (G4S)n, wherein n = 1-10, e.g. 1, 2 or 3; and / or the C-terminal linker is selected from the C-terminal contiguous fragment of the chain in the protein from which the chain is derived; the C-terminal fragment of the heavy chain constant region CH1 (e.g. when it is N-terminally connected to a heavy chain variable region), the C-terminal fragment of the light chain constant region (e.g. when it is N-terminally connected to a light chain variable region), or (G4S)n, wherein n = 1-10, e.g. 1, 2 or 3; Preferably, wherein, the PD1 chain comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at its N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13), ERRAE SC (SEQ ID NO: 21) or ERRGEC (SEQ ID NO: 22) at its C-terminus; or the PD1 chain comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at its N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13), ERRAE SC (SEQ ID NO: 21) or ERRGEC (SEQ ID NO: 22) at its C-terminus; or the PDL1 chain comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at its N-terminus and a C-terminal linker VKVNAPYNK (SEQ ID NO: 51) or VKVNAPYNKSC (SEQ ID NO: 53) at its C-terminus; or the PDL1 chain comprises an N-terminal linker ASTKGP (SEQ ID NO: 31) at its N-terminus and a C-terminal linker VKVNAPYNK (SEQ ID NO: 51) or VKVNAPYNKSC (SEQ ID NO: 53) at its C-terminus; or the PDL2 chain comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at its N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) or VKASYRKGEC (SEQ ID NO: 56) at its C-terminus; or the PDL2 chain comprises an N-terminal linker ASTKGP (SEQ ID NO: 31) at its N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) or VKASYRKSC (SEQ ID NO: 55) at its C-terminus.

5. The polypeptide complex of any one of claims 1-4, wherein the modified PD1 chain comprising an N-terminal and a C-terminal linker comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2-7; the modified PDL1 chain comprising an N-terminal and a C-terminal linker comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 15-18; or the modified PDL2 chain comprising an N-terminal and a C-terminal linker comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 24-27.

6. The polypeptide complex of any one of claims 1-5, wherein the combination of the first domain and the second domain is selected from the group consisting of, respectively 7. The polypeptide complex of any one of claims 1-6, wherein (i). the first domain is a PD1 chain and the second domain is a PDL1 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 8 and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 19; (ii). the first domain is a PDL1 chain and the second domain is a PD1 chain, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 19 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 8; (iii). the first domain is a PD1 chain and the second domain is a PDL2 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9 and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28; (iv). the first domain is a PDL2 chain and the second domain is a PD1 chain, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9; (v). the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 10 and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 20; (vi). the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises the amino acid sequence set forth in SEQ ID NO: 20 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 10; (vii). the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 11 and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 29; or (viii). the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 29 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO:

11.

8. The polypeptide complex of any one of claims 1-7, wherein the combination of the first domain and the second domain is selected from the group consisting of, respectively 9. The polypeptide complex of any one of claims 1-8, wherein (i). the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; (ii). the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2; (iii). the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24; (iv). the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; (v). the first domain is a PD1 chain, the second domain is a PDL1 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17; (vi). the first domain is a PDL1 chain, the second domain is a PD1 chain, and the PDL1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5; (vii). the first domain is a PD1 chain, the second domain is a PDL2 chain, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27; or (viii). the first domain is a PDL2 chain, the second domain is a PD1 chain, and the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

7.

10. The polypeptide complex of claim 1, wherein the first domain comprises a CD47 chain, and the second domain comprises a domain capable of interacting with the CD47 chain, such as a SIRPa chain; the first domain comprises a domain capable of interacting with the CD47 chain, such as a SIRPa chain, and the second domain comprises a CD47 chain; the first domain comprises a SIRPa chain, and the second domain comprises a domain capable of interacting with the SIRPa chain, such as a CD47 chain; or the first domain comprises a domain capable of interacting with the SIRPa chain, such as a CD47 chain, and the second domain comprises a SIRPa chain.

11. The polypeptide complex of claim 10, wherein the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32, or is a modified CD47 chain having a cysteine mutation (i.e., mutated to cysteine) to create an interchain disulfide bond and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid such as S, K, D, Q, or E) relative to the parent CD47 chain set forth in SEQ ID NO: 32, preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or binding of the CD47 chain to the SIRPa chain; or the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, or is a modified SIRPa chain having a cysteine mutation (i.e., mutated to cysteine) to create an interchain disulfide bond and / or an N-glycosylation site mutation (i.e., mutated to a non-glycosylated amino acid) relative to the parent SIRPa chain set forth in SEQ ID NO: 57, preferably the N-glycosylation site mutation does not affect the formation of its interchain disulfide bond and / or binding of the SIRPa chain to the CD47 chain.

12. The polypeptide complex of any one of claims 1, 10, or 11, wherein the modified CD47 chain has one or more amino acid residue mutations selected from 5K, 16D, 29C, 32S, 46C, 55Q, 93E / K, and 104C, or has a combination of mutations 5K, 16D, 32S, 55Q, and 93E / K and optionally one or more other mutations, such as 29C, 46C, and / or 104C, wherein the modified CD47 chain mutation sites are sequentially numbered sites relative to the sequence of SEQ ID NO: 32; Preferably, the modified CD47 chain has the following combination of mutations: N5K, N16D, N32S, N55Q, N93E, E104C; N5K, N16D, N32S, N55Q, N93K, E104C; N5K, N16D, E29C, N32S, N55Q, N93E; N5K, N16D, E29C, N32S, N55Q, N93K; N5K, N16D, N32S, D46C, N55Q, N93E; or N5K, N16D, N32S, D46C, N55Q, N93K; More preferably, the modified CD47 chain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 45-50; or the modified SIRPa chain has one or more amino acid residue mutations selected from 53C, 69C and / or 98C, wherein the modified SIRPa chain mutation site is the ordinal number site relative to sequence SEQ ID NO: 57; Preferably, the modified SIRPa chain has the following mutations: K53C; R69C; or S98C; More preferably, the modified SIRPa chain comprises the amino acid sequence of any one of SEQ ID NOs: 64-66.

13. The polypeptide complex of any one of claims 10-12, wherein the CD47 chain or SIRPa chain further comprises a linker, such as an N-terminal linker at the N-terminus and / or a C-terminal linker at the C-terminus, wherein the N-terminal linker is selected from the N-terminal contiguous fragment of the chain in the protein from which the chain is derived, the N-terminal fragment of a heavy chain constant region (e.g. when it is N-terminally connected to a heavy chain variable region), the N-terminal fragment of a light chain constant region (e.g. when it is N-terminally connected to a light chain variable region), or (G4S)n, wherein n = 1-10, such as 1, 2 or 3; and / or the C-terminal linker is selected from the C-terminal contiguous fragment of the chain in the protein from which the chain is derived; the C-terminal fragment of a heavy chain constant region CH1 (e.g. when it is N-terminally connected to a heavy chain variable region), the C-terminal fragment of a light chain constant region (e.g. when it is N-terminally connected to a light chain variable region), or (G4S)n, wherein n = 1-10, such as 1, 2 or 3; Preferably, wherein the CD47 chain comprises an N-terminal linker (G4S)1(SEQ ID NO: 76) at its N-terminus and a C-terminal linker AKEPKSA (SEQ ID NO: 67) or GE at its C-terminus; or the SIRPa chain comprises an N-terminal linker (G4S)1(SEQ ID NO: 76) at its N-terminus and a C-terminal linker EPKSA (SEQ ID NO: 68) or GE at its C-terminus.

14. The polypeptide complex of any one of claims 10-13, wherein the modified CD47 chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 33-44; or the modified SIRPa chain comprising N-terminal and C-terminal linkers comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 58-63.

15. The polypeptide complex of any one of claims 10-14, wherein the combination of the first domain and the second domain is selected from the group consisting of, respectively 16. The polypeptide complex of any one of claims 10-15, wherein the combination of the first and second domains is selected from the combinations, respectively, wherein (i). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 45 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64; (ii). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 46 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64; (iii). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 47 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 65; (iv). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 48 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 65; (v). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 49 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66; (vi). the first domain is a CD47 chain and the second domain is a SIRPa chain, and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 50 and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66; (vii). the first domain is a SIRPa chain and the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 45; (viii). the first domain is a SIRPa chain and the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 64 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 46; (ix). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 65 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 47; (x). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 65 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 48; (xi). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 49; or (xii). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO:

50.

17. The polypeptide complex of any one of claims 10-16, wherein the combination of the first domain and the second domain is selected from the group consisting of, respectively 18. The polypeptide complex of any one of claims 10-17, wherein the combination of the first and second domains is selected from the combinations, respectively, of: (i). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; (ii). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; (iii). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; (iv). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; (v). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37 and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; (vi). the first domain is a CD47 chain, the second domain is a SIRPa chain, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; (vii). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39; (viii). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40; (ix). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; (x). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42; (xi). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43; or (xii). the first domain is a SIRPa chain, the second domain is a CD47 chain, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63, and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

44.

19. The polypeptide complex of any one of claims 1-18, wherein the first domain is further operably linked to an Fc region, wherein the first antigen binding moiety comprises or consists of: a first polypeptide chain or heavy chain comprising, from N- to C-terminus, a heavy chain variable region VH - a first domain (Domain 1) - an Fc region, or a heavy chain variable region VH - a first domain (Domain 1) - an Fc region; a second polypeptide chain or light chain comprising, from N- to C-terminus, a light chain variable region VL - a second domain (Domain 2), or a light chain variable region VL - a second domain (Domain 2); wherein the first domain Domain 1 and the second domain Domain 2 are capable of interacting to form a dimer, and are as defined in any one of claims 1-18. ​ ​ ​ 20. The polypeptide complex of claim 19, wherein the Fc region is or is from an IgG Fc, e.g., is or is from an Fc region of (human) IgGl, IgG2, IgG3, or IgG4, e.g., the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or more, thereto.

21. The polypeptide complex of any one of claims 1-20, which is multispecific, e.g., bispecific, e.g., a bispecific antibody, and further comprises a second antigen binding moiety.

22. The polypeptide complex of claim 21, wherein the second antigen binding moiety comprises: a third polypeptide comprising, from N- to C-terminal, VH2 operably linked to a third domain comprising or being CH1, and a fourth polypeptide comprising, from N- to C-terminal, VL2 operably linked to a fourth domain comprising or being CL.

23. The polypeptide complex of claim 22, wherein the CH1 is a human IgG CH1, e.g., a human IgGl CH1, a human IgG2 CH1, a human IgG3 CH1, or a human IgG4 CH1, e.g., the CH1 region comprises or consists of the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or 100%, thereto.

24. The polypeptide complex of claim 22 or 23, wherein the CL is a human Kappa light chain CL region or a human Lamda light chain CL region, e.g., the CL region comprises or consists of the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 98%, 99% or 100%, thereto.

25. The polypeptide complex of any one of claims 1-24, comprising or consisting of a first antigen binding moiety and a second antigen binding moiety, wherein the first antigen binding moiety comprises or consists of: a first polypeptide chain or first heavy chain comprising, from N-terminal to C-terminal, a heavy chain variable region VH1 - a first domain (Domain 1) - a first Fc region, or a heavy chain variable region VH1 - a first domain (Domain 1) - a first Fc region; a second polypeptide chain or first light chain comprising, from N-terminal to C-terminal, a light chain variable region VL1 - a second domain (Domain 2), or a light chain variable region VL1 - a second domain (Domain 2); and the second antigen binding moiety comprises: a third polypeptide chain or second heavy chain comprising, from N-terminal to C-terminal, a heavy chain variable region VH2 - CH1 - a second Fc region, or a heavy chain variable region VH2 - CH1 - a second Fc region; ​ the fourth polypeptide chain or the second light chain comprises from N-terminus to C- terminus a light chain variable region VL2-CL, or is a light chain variable region VL2-CL; wherein the first antigen binding moiety specifically binds to a first antigen and the second antigen binding moiety specifically binds to a second antigen, the first domain (Domain 1) and the second domain (Domain 2) are capable of interacting to form a dimer, and are as defined in any one of claims 1-18.

26. The polypeptide complex of claim 25, wherein the first and second Fc regions are or are from an IgG Fc, e.g., from an Fc region of (human) IgG1, IgG2, IgG3, or IgG4, e.g., the first and second Fc regions are different, e.g., they each comprise a Knob-into-hole mutation, e.g., the first Fc region comprises a Knob mutation, e.g., T366W, and the second Fc region comprises a Hole mutation, e.g., T366S, L368A, and Y407V; optionally, the second Fc region further comprises H445R and Y446F.

27. The polypeptide complex of claim 26, wherein one Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78, and the other Fc region polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79; one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 78, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 79; or one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 78 and comprises the mutation T366W, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 79 and comprises the mutations T366S, L368A, Y407V, H445R, and Y446F.

28. The polypeptide complex of any one of claims 1-27, wherein the VH1 and VL1 of the first antigen binding moiety form a first antigen binding site that specifically binds to a first antigen; and / or the VH2 and VL2 of the second antigen binding moiety form a second antigen binding site that specifically binds to a second antigen; wherein the first and second antigen binding sites specifically bind to the first and second antigens, respectively, wherein the first and second Fc regions are or are from an IgG Fc, e.g., from an Fc region of (human) IgG1, IgG2, IgG3, or IgG4, e.g., the first and second Fc regions are different, e.g., they each comprise a Knob-into-hole mutation, e.g., the first Fc region comprises a Knob mutation, e.g., T366W, and the second Fc region comprises a Hole mutation, e.g., T366S, L368A, and Y407V; optionally, the second Fc region further comprises H445R and Y446F. The first or second antigen is selected from a tumor-specific antigen or a tumor-associated antigen, for example the tumor-associated antigen is an immune checkpoint molecule, for example the antigen is selected from C-Met, EGFR, HER3, 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MFI2, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD1lc, CD123, CD138, CD142, CD147, CD166, CD19, CD19,CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45 (PTPRC), CD46, CD47, CD49D (ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH11, CDllb, CEA, CEACAM5, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHl, FGFR2, FGFR3, FLT3, FOLR-a, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pCAD, P-Cadherin, PDGFRA, PDK1, PDL1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAhlg, PSMA, PTK7, P-cadherin, RNF43, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, ADAM9, epidermal growth factor, short proteoglycans, mesothelin, sodium phosphate cotransporter 2B, Claudin 18.2, endothelin receptor, mucins (such as Mucin 1 and Mucin 16), guanylate cyclase C, integrin a4b7, integrin avb6, trophoblast glycoprotein, and tissue factor.

29. The polypeptide complex of claim 28, wherein the antigen-binding site that specifically binds C-met comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein (i) the VH comprises 3 HCDR1, HCDR2, and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 83; and the VL comprises 3 LCDR1, LCDR2, and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 84; (ii) the VH comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3, and the VL comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 144; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 145, H the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 146, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 147, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 148, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 149; (iii) the VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83 and the VL comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84; or (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 83 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 84; or the antigen-binding site that specifically binds EGFR comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein (i) the VH comprises 3 HCDR1, HCDR2, and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 85; and the VL comprises 3 LCDR1, LCDR2, and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 86; (ii) the VH comprises 3 complementarity determining regions from a heavy chain variable region (HCDRs), HCDR1, HCDR2, and HCDR3, and the VL comprises 3 complementarity determining regions from a light chain variable region (LCDRs), LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 150; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 151, H the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 152, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 153, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 154, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 155; and (iii) the VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 85 and the VL comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86; or (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 85 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:

86. (iii) the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 85 or consists of the amino acid sequence and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86 or consists of the amino acid sequence; or (iv) the VH comprises the amino acid sequence of SEQ ID NO: 85 or consists of the amino acid sequence and the VL comprises the amino acid sequence of SEQ ID NO: 86 or consists of the amino acid sequence; or the antigen binding site that specifically binds to HER3 comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein (i) the VH comprises 3 heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 87 and the VL comprises 3 light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 88; (ii) the VH comprises 3 heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 from a heavy chain variable region and the VL comprises 3 light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 from a light chain variable region, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 156; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 157, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 158, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 159, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 160, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 161; (iii) the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 87 or consists of the amino acid sequence and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88 or consists of the amino acid sequence; or (iv) the VH comprises the amino acid sequence of SEQ ID NO: 87 or consists of the amino acid sequence and the VL comprises the amino acid sequence of SEQ ID NO: 88 or consists of the amino acid sequence; or the antigen binding site that specifically binds to PDL1 comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein (i) the VH comprises 3 heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 87 and the VL comprises 3 light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 88; (ii) the VH comprises 3 heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 from a heavy chain variable region and the VL comprises 3 light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 from a light chain variable region, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 156; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 157, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 158, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 159, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 160, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 161; (iii) the VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 87 or consists of the amino acid sequence and the VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88 or consists of the amino acid sequence; or (iv) the VH comprises the amino acid sequence of SEQ ID NO: 87 or consists of the amino acid sequence and the VL comprises the amino acid sequence of SEQ ID NO: 88 or consists of the amino acid sequence; or (i) the VH comprises 3 HCDR1, HCDR2, and HCDR3 from a heavy chain variable region as set forth in SEQ ID NO: 87, and the VL comprises 3 LCDR1, LCDR2, and LCDR3 from a light chain variable region as set forth in SEQ ID NO: 88; (ii) the VH comprises 3 complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises 3 complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 164; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 165, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 166, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 167, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 168, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 169; (iii) the VH comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 162 and the VL comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 163; or (iv) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 162 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:

163.

30. The polypeptide complex of any one of claims 1-29, wherein (a) the VH1 and VL1 of the first antigen binding moiety make up an antigen binding site that specifically binds HER3 and the VH2 and VL2 of the second antigen binding moiety make up an antigen binding site that specifically binds EGFR, and the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain; the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain; (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C, and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus compared to the parent PDL2 chain, and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D, and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus compared to the parent PD1 chain; (iii) the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; or (iv) the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9; Preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 73; More preferably, wherein the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73; or the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light (b) the VH1 and VL1 of the first antigen binding moiety make up an antigen binding site that specifically binds to HER3 and the VH2 and VL2 of the second antigen binding moiety make up an antigen binding site that specifically binds to EGFR, and the first domain is a SIRPa chain and the second domain is a CD47 chain, wherein (i) the SIRPa chain comprises the mutation S98C compared to the parental SIRPa chain and the CD47 chain comprises the mutation combination N5K, N16D, N32S, D46C, N55Q, N93K compared to the parental CD47 chain; (ii) the SIRPa chain comprises the mutation S98C compared to the parental SIRPa chain and comprises an N-terminal linker G4S at the N-terminus and a C-terminal linker GE at the C-terminus and the CD47 chain comprises the mutation combination N5K, N16D, N32S, D46C, N55Q, N93K compared to the parental CD47 chain and comprises an N-terminal linker G4S at the N-terminus and a C-terminal linker GE at the C-terminus (iii) the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 and the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

44. (iv) the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 66 and the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 50; preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 136, the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 141; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 73; more preferably, wherein the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 136, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 141; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

73. the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 131; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

73.

31. The polypeptide complex of any one of claims 1-29, wherein the VH1 and VL1 of the first antigen binding moiety make up an antigen binding site that specifically binds EGFR, and the VH2 and VL2 of the second antigen binding moiety make up an antigen binding site that specifically binds HER3, and the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain; (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus, and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus; (iii) the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25 and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; or (iv) the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28 and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO: 9; Preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain as set forth below, wherein the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 134, the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 140; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 132; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

73. the second heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 132; and more preferably wherein the first heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 134, the first light chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 140; the second heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 132; and the second light chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO:

75.

32. The polypeptide complex of any one of claims 1-29, wherein the VH1 and VL1 of the first antigen binding moiety make up an antigen binding site that specifically binds C-met, and the VH2 and VL2 of the second antigen binding moiety make up an antigen binding site that specifically binds HER3, and the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain; (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain, and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain, and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus; (iii) the PDL2 chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 25, and the PD1 chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 3; or (iv) the PDL2 chain comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the PD1 chain comprises an amino acid sequence as set forth in SEQ ID NO: 9; preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain as set forth below, wherein the first heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence as set forth in SEQ ID NO: 138, the first light chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 140; the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 138; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 132; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 75; Preferably, wherein the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 138, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

75.

33. The polypeptide complex of any one of claims 1-29, wherein the VH1 and VL1 of the first antigen binding moiety make up an antigen binding site that specifically binds HER3, and the VH2 and VL2 of the second antigen binding moiety make up an antigen binding site that specifically binds C-met, and the first domain is a PDL2 chain and the second domain is a PD1 chain, wherein (i) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain; (ii) the PDL2 chain comprises the mutation combination N17D, N44D, V88C compared to the parental PDL2 chain, and comprises an N-terminal linker RTVAAP (SEQ ID NO: 30) at the N-terminus and a C-terminal linker VKASYRK (SEQ ID NO: 54) at the C-terminus; and the PD1 chain comprises the mutation combination N15S, N24S, V30C, N40S, C59S, N82D compared to the parental PD1 chain, and comprises an N-terminal linker DRPWNP (SEQ ID NO: 12) at the N-terminus and a C-terminal linker ERRAE (SEQ ID NO: 13) at the C-terminus; (iii) the PDL2 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and the PD1 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; or (iv) the PDL2 chain comprises the amino acid sequence set forth in SEQ ID NO: 28, and the PD1 chain comprises the amino acid sequence set forth in SEQ ID NO:

9. Preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 133; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 71; More preferably, the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 139; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 133; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

71.

34. The polypeptide complex of any one of claims 1-29, wherein the VH1 and VL1 of the first antigen binding moiety constitute an antigen binding site that specifically binds HER3, and the VH2 and VL2 of the second antigen binding moiety constitute an antigen binding site that specifically binds PDL1, and the first domain is a CD47 chain and the second domain is a SIRPa chain, wherein (i) the CD47 chain comprises the mutation combination N5K, N16D, E29C, N32S, N55Q, N93E compared to the parental CD47 chain, and the SIRPa chain comprises the mutation R69C compared to the parental SIRPa chain; (ii) the CD47 chain comprises the mutation combination N5K, N16D, E29C, N32S, N55Q, N93E compared to the parental CD47 chain, and comprises an N-terminal linker G4S at the N-terminus and an C-terminal linker AKEPKSA (Seq ID NO: 67) at the C-terminus; and the SIRPa chain comprises the mutation R69C compared to the parental SIRPa chain, and comprises an N-terminal linker G4S at the N-terminus and an C-terminal linker EPKSA (Seq ID NO: 68) at the C-terminus; (iii) the CD47 chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, and the SIRPa chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; or (iv) the CD47 chain comprises the amino acid sequence set forth in SEQ ID NO: 47, and the SIRPa chain comprises the amino acid sequence set forth in SEQ ID NO: 65; Preferably, the polypeptide complex comprises or consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 172, the first light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 173; the second heavy chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 170; and the second light chain comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 171; More preferably, the first heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 172, the first light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 173; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 170; and the second light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

171.

35. A nucleic acid encoding the polypeptide complex of any one of claims 1-34, or any one or more chains thereof.

36. An expression vector comprising the nucleic acid of claim 35.

37. A host cell comprising the nucleic acid of claim 35 or the expression vector of claim 36.

38. A method of making the polypeptide complex of any one of claims 1-34, comprising a) introducing nucleic acids encoding each chain of the polypeptide complex into a host cell; b) expressing and assembling the antibody or antigen binding fragment thereof in vivo in the host cell; Optionally, the polypeptide complex is purified, e.g., by Protein A purification.

39. An immunoconjugate comprising the polypeptide complex of any one of claims 1-34.

40. A pharmaceutical composition comprising the polypeptide complex of any one of claims 1-34 or the immunoconjugate of claim 39, and a pharmaceutically acceptable carrier or excipient.

41. A pharmaceutical combination comprising the polypeptide complex of any one of claims 1-34 or the immunoconjugate of claim 39 or the pharmaceutical composition of claim 40, and a further therapeutic agent, e.g., wherein the further therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a small molecule drug, an angiogenesis inhibitor, an immunomodulatory agent, or another anti-tumor antibody.

42. Use of the polypeptide complex of any one of claims 1-34 or the immunoconjugate of claim 39 or the pharmaceutical composition of claim 40 in the manufacture of a medicament for the treatment and / or prevention of a disease or condition.

43. The polypeptide complex of any one of claims 1-34 or the immunoconjugate of claim 39 or the pharmaceutical composition of claim 40 for use in the treatment and / or prevention of a disease or condition.

44. A method of treating and / or preventing a disease or condition comprising administering to a subject in need thereof the polypeptide complex of any one of claims 1-34 or the immunoconjugate of claim 39 or the pharmaceutical composition of claim 40.

45. The use of claim 42, or the polypeptide complex or immunoconjugate or pharmaceutical composition of claim 43, or the method of claim 44, wherein the disease or condition is selected from a proliferative disease and / or condition, a metabolic disease and / or condition, an inflammatory disease and / or condition, and a neurodegenerative disease and / or condition, e.g., the disease and / or condition is a tumor, e.g., a solid tumor or a hematological tumor, e.g., a cancer, e.g., the tumor or cancer expresses or overexpresses a tumor-specific antigen or a tumor-associated antigen, e.g., the tumor-associated antigen is an immune checkpoint molecule.

46. The use of claim 42 or 45, or the polypeptide complex or immunoconjugate or pharmaceutical composition of claim 43 or 45, or the method of claim 44 or 5, wherein the polypeptide complex or immunoconjugate or pharmaceutical composition is administered in combination with one or more additional therapeutic methods, wherein the additional therapeutic methods are selected from radiation therapy, chemotherapy, immunotherapy, or a combination thereof; or the polypeptide complex or immunoconjugate or pharmaceutical composition is administered in combination with one or more other therapeutic agents, wherein the other therapeutic agents are selected from a chemotherapeutic agent, a cytotoxic agent, a small molecule drug, an angiogenesis inhibitor, an immunomodulatory agent, or another anti-tumor antibody.

47. A method of detecting the presence of an antigen bound by a polypeptide complex in a biological sample, the method contacting a biological sample with the polypeptide complex of any one of claims 1-34 under conditions in which it binds thereto, and detecting whether a complex is formed between the polypeptide complex and the antigen, wherein formation of a complex indicates the presence of the antigen.

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