EA045275B1 - ANTI-PD-L1/ANTI-4-1BB BISPECIFIC ANTIBODIES AND THEIR APPLICATIONS - Google Patents

Description

BACKGROUND OF THE INVENTION

Programmed death protein ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type 1 transmembrane protein that is believed to play a major role in suppression immune system during specific events such as pregnancy, tissue allografts, autoimmune diseases and other disease states such as hepatitis. The binding of PD-L1 to PD-1 or B7.1 conveys an inhibitory signal that reduces the proliferation of CD8+ T cells in lymph nodes, and in addition to this, PD-1 is also able to control the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis, which is further mediated by lower regulation of the Bcl-2 gene.

It has been shown that up-regulation of PD-L1 may allow cancer to evade the host immune system. An analysis of tumor samples taken from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and an increased risk of death. Many PD-L1 inhibitors are in development as immuno-oncology therapies and are showing good results in clinical trials.

4-1BB is a member of the TNF receptor superfamily (TNFRSF) and is a co-stimulatory molecule that is expressed following activation of immune cells, both the innate and adaptive immune systems. 4-1BB plays an important role in modulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Many other studies have shown that activation of 4-1BB enhances the immune response to eliminate tumors in mice. Therefore, 4-1BB is suggested to be a promising target molecule in cancer immunology. Despite its antitumor efficacy, anti-4-1BB antibody causes severe liver toxicity when used clinically.

Brief Disclosure of the Present Invention

The present invention provides an anti-PD-L1/anti-4-1BB bispecific antibody capable of effectively blocking interactions between PD-L1 and its receptor PD-1 and between 4-1BB and its ligand. The bispecific antibody may have high binding affinity to both PD-L1 protein (eg, human PD-L1 protein) and 4-1BB protein (eg, human 4-1BB protein).

The anti-PD-L1/anti-4-1BB bispecific antibody may comprise an anti-PD-L1 antibody or an antigen-binding fragment thereof as a PD-L1-targeting moiety that is capable of specifically recognizing and/or binding to the PD-L1 protein, and anti -4-1BB antibody or antigen-binding fragment thereof as a 4-1BB-targeting moiety that is capable of specifically recognizing and/or binding to the 4-1BB protein.

The anti-PD-L1/anti-4-1BB bispecific antibody may contain an anti-PD-L1 antibody or an antigen binding fragment thereof as a PD-L1-targeting moiety.

In an embodiment, an anti-PD-L1 antibody or fragment thereof contained in a bispecific antibody may specifically bind to the immunoglobulin C (IgC) domain of a PD-L1 protein (eg, human PD-L1). In some embodiments, the IgC domain consists of amino acid residues 133-225 of the human PD-L1 protein. In some embodiments, the anti-PD-L1 antibody or fragment thereof can bind to at least one of amino acid residues Y134, K162, and N183 of the human PD-L1 protein. In some embodiments, the anti-PD-L1 antibody or fragment thereof is incapable of binding to the immunoglobulin V (IgV) domain of the PD-L1 protein, and, for example, the IgV domain consists of amino acid residues 19-127 of the human PD-L1 protein. For example, the human PD-L1 protein may be selected, without limitation, from the group consisting of proteins listed in GenBank under accession number NP_001254635.1, NP_001300958.1, NP_054862.1, etc. These anti-PD-L1 antibodies may be useful for therapeutic purposes such as treating various types of cancer, etc., and can also be used for diagnostic and prognostic purposes. In an embodiment, the anti-PD-L1 antibody or fragment thereof is capable of specificity for the human PD-L1 protein.

The anti-PD-L1/anti-4-1BB bispecific antibody comprises an anti-PD-L1 antibody or an antigen binding fragment thereof and an anti-4-1BB antibody or an antigen binding fragment thereof, wherein the anti-PD-L1 antibody or an antigen binding fragment thereof is capable of specifically binding with the immunoglobulin C (IgC) domain of the human programmed death protein ligand 1 (PD-L1) protein, where the IgC domain consists of amino acid residues 133-225, the anti-PD-L1 antibody or antigen binding fragment thereof contains a CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 3, CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 5, 262, 263, 264, 265, 266 and 267, CDR1 VL having the amino acid sequence of SEQ ID NOs: 6, 268 and 269, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8, 270 , 271 and 272, and the anti-4-1BB antibody or antigen binding fragment thereof comprises a VH CDR1 having the amino acid sequence

- 1 045275 selected from the group consisting of SEQ ID NOs: 10 and 11, CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16 and 17, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20.

In an embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof comprises a CDR1 VH having the amino acid sequence of SEQ ID NO: 1, a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 3, CDR3 VH, having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 5, CDR1 VL having the amino acid sequence of SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8.

In an embodiment, the αntu-PD-L1 antibody or antigen binding fragment thereof is capable of binding to at least one of amino acid residues Y134, K162, or N183 of the PD-L1 protein.

In an embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof is capable of binding to amino acid residues Y134, K162, and N83 of the PD-L1 protein.

In an embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof is incapable of binding to the immunoglobulin V (IgV) domain of the PD-L1 protein, where the IgV domain consists of amino acid residues 19-127.

In an embodiment, the anti-PD-L1/anti-4-1BB bispecific antibody activates 4-1BB signaling or an immune response depending on the PD-L1 expressed on cell surfaces.

In an embodiment, each of the anti-PD-L1 antibody or antigen binding fragment thereof and the anti-4-1BB antibody or antigen binding fragment thereof is independently a chimeric antibody, a humanized antibody, or a fully human antibody.

The anti-PD-L1/anti-4-1BB bispecific antibody comprises an anti-PD-L1 antibody or an antigen binding fragment thereof and an anti-4-1BB antibody or an antigen binding fragment thereof, wherein the anti-PD-L1 antibody or an antigen binding fragment thereof comprises (1 ) CDR1 VH having the amino acid sequence of SEQ ID NO: 1, (2) CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 3, (3) CDR3 VH having an amino acid sequence selected from the group , consisting of SEQ ID NOs: 4, 5, 262, 263, 264, 265, 266 and 267, (4) CDR1 VL having the amino acid sequence of SEQ ID NOs: 6, 268 and 269, (5) CDR2 VL having the amino acid the sequence SEQ ID NO: 7, and (6) CDR3 VL having the amino acid sequence SEQ ID NO: 8, 270, 271 and 272; and the anti-4-1BB antibody or antigen binding fragment thereof comprises (i) a CDR1 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11, (ii) a CDR2 VH having an amino acid sequence selected from the group, consisting of SEQ ID NOs: 12 and 13, (iii) CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16 and 17, (iv) CDR1 VL having the amino acid sequence of SEQ ID NO : 18, (v) a VL CDR2 having the amino acid sequence of SEQ ID NO: 19, and (vi) a VL CDR3 having the amino acid sequence of SEQ ID NO: 20.

The anti-PD-LI antibody or fragment thereof may comprise (1) a CDR1 VH having the amino acid sequence of SEQ ID NO: 1;

(2) a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 3;

(3) a CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 262, 263, 264, 265, 266 and 267;

(4) CDR1 VL having the amino acid sequence of SEQ ID NO: 6, 268 and 269;

(5) CDR2 VL having the amino acid sequence of SEQ ID NO: 7; and (6) CDR3 VL having the amino acid sequence of SEQ ID NOs: 8, 270, 271 and 272.

For example, an anti-PD-L1 antibody or fragment thereof may comprise a CDR1 VH having the amino acid sequence of SEQ ID NO: 1, a CDR2 VH having the amino acid sequence of SEQ ID NO: 2 or 3, a CDR3 VH having the amino acid sequence of SEQ ID NO: 4 , 5, 262, 263, 264, 265, 266 or 267, CDR1 VL having the amino acid sequence of SEQ ID NO: 6, 268 or 269, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL,

- 2 045275 having the amino acid sequence SEQ ID NO: 8, 270, 271 or 272.

In an embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof comprises a CDR1 VH having the amino acid sequence of SEQ ID NO: 1, a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 3, CDR3 VH, having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 5, CDR1 VL having the amino acid sequence of SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8.

The anti-PD-L1/anti-4-1BB bispecific antibody may contain an anti-4-1BB antibody or an antigen binding fragment thereof as a 4-1BB-targeting moiety. In an embodiment, the anti-4-1BB antibody or fragment thereof may specifically bind to a 4-1BB protein (eg, human 4-1BB).

An anti-4-1BB antibody or an antigen-binding fragment thereof is capable of enhancing the immune response and/or treating a tumor (cancer) in a mammal. An anti-4-1BB antibody or antigen-binding fragment thereof is characterized by localization and/or activation only in the tumor microenvironment (TME) and/or significantly reduced liver toxicity compared to pre-existing anti-4-1BB antibodies, while maintaining the effectiveness of enhancing the immune response and /or tumor treatment.

For example, the human 4-1BB protein may be selected without limitation from the group consisting of proteins submitted to NCBI under accession number NP_001552, etc. These anti-4-1BB antibodies may be useful for therapeutic purposes such as treatment of various types of cancer, etc., and may also be used for diagnostic and prognostic purposes.

In an embodiment, the anti-4-1BB antibody or fragment thereof is capable of specificity for human 4-1BB protein. The Anit-4-1BB antibody or fragment thereof may comprise (i) a CDR1 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11;

(ii) a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13;

(iii) a CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16 and 17;

(iv) CDR1 VL having the amino acid sequence of SEQ ID NO: 18;

(v) CDR2 VL having the amino acid sequence of SEQ ID NO: 19; and (vi) CDR3 VL having the amino acid sequence of SEQ ID NO: 20.

For example, an anti-4-1BB antibody or fragment thereof may comprise a CDR1 VH having the amino acid sequence of SEQ ID NO: 10 or 11, a CDR2 VH having the amino acid sequence of SEQ ID NO: 12 or 13, a CDR3 VH having the amino acid sequence of SEQ ID NO: : 14, 15, 16 or 17, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20.

In an embodiment, each of the anti-PD-L1 antibody or antigen binding fragment thereof and the anti-4-1BB antibody or antigen binding fragment thereof is independently a chimeric antibody, a humanized antibody, or a fully human antibody.

In an embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof and the anti-4-1BB antibody or antigen binding fragment thereof are humanized antibodies.

In an embodiment, the αanti-PD-L1 antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 and 104, or a polypeptide having at least 90% sequence identity with the amino acid a sequence selected from the group consisting of SEQ ID NOs: 103 and 104.

In an embodiment, the anti-PD-L1 antibody or antigen binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105 and 106, or a peptide having at least 90% sequence identity with the amino acid a sequence selected from the group consisting of SEQ ID NOs: 105 and 106.

In an embodiment, the anti-4-1BB antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23 and 24, or a polypeptide having a sequence identity of at least 90 % with an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24.

In an embodiment, the anti-4-1BB antibody or antigen binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26, or a peptide having a sequence identity of at least 90 % with an amino acid sequence selected from the group consisting of SEQ ID NO: 25 and 26.

In an embodiment, the anti-PD-L1/anti-4-1BB bispecific antibody activates 4-1BB signaling depending on PD-L1 expressed on cell surfaces.

In an embodiment, the anti-PD-L1/anti-4-1BB bispecific antibody is in the form of an IgG-scFv.

The anti-PD-L1/anti-4-1BB bispecific antibody may contain a heavy component containing an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41 and 43, and a light component comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42 and 44.

According to another embodiment, the present invention relates to a pharmaceutical composition containing a bispecific antibody as described above. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition can be used for the treatment and/or prevention of cancer.

In another embodiment, the present invention provides a method for treating and/or preventing cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a bispecific antibody or pharmaceutical composition. The method may further include the step of identifying a subject in need of cancer treatment and/or prevention prior to the administration step.

According to another embodiment, the present invention relates to the use of a bispecific antibody or pharmaceutical composition for the treatment and/or prevention of cancer. According to another embodiment, the present invention relates to the use of a bispecific antibody for the preparation of a pharmaceutical composition for the treatment and/or prevention of cancer.

With respect to the pharmaceutical compositions, methods and/or uses disclosed herein, the cancer may be a solid cancer or a blood cancer, preferably a solid cancer.

In another embodiment, the present invention provides a composition for detecting PD-L1, 4-1BB, or both in a biological sample, the composition comprising a bispecific antibody. In another embodiment, the present invention provides a method for detecting PD-L1, 4-1BB, or both in a biological sample, the method comprising contacting the biological sample with a bispecific antibody and detecting (measuring) an antigen-antibody reaction (binding). between the bispecific antibody and PD-L1, 4-1BB, or both.

The detection method may, after the detection step, further comprise determining whether PD-L1, 4-1BB, or both is present in the biological sample when an antigen-antibody response is detected, and/or whether PD-L1, 4-1BB, or both in a biological sample when no antigen-antibody reaction is detected.

According to another embodiment, the present invention provides a pharmaceutical composition for diagnosing a disease associated with PD-L1, 4-1BB, or both, the composition comprising a bispecific antibody. In another embodiment, the present invention relates to the use of a bispecific antibody for diagnosing a disease associated with PD-L1, 4-1BB, or both.

In another embodiment, the present invention provides a method for diagnosing a disease associated with PD-L1, 4-1BB, or both, the method comprising contacting a biological sample taken from a patient with a bispecific antibody and detecting an antigen-antibody reaction or measuring the level of antigen-antibody reaction in a biological sample. In some embodiments, the method may further comprise contacting a normal sample with a bispecific antibody and measuring the level of antigen-antibody reaction in the normal sample. In addition, the method may further include comparing the level of antigen-antibody reaction in the biological sample and in the normal sample after the measurement step. In addition, after the detection step or comparison step, the method may further include identifying the patient as having a disease associated with PD-L1, 4-1BB, or both, when an antigen-antibody reaction is detected in the biological sample or the level of the antigen-antibody reaction is detected. the antibody in a biological sample is higher than that in a normal sample.

Disease associated with PD-L1, 4-1BB, or both may be due to activation (eg, abnormal activation or overactivation) and/or overproduction (overexpression) of PD-L1, 4-1BB, or both and another. For example, the disease may be cancer, as described above.

In an embodiment, the present invention provides a polynucleotide encoding a bispecific antibody. In particular, according to an embodiment, the present invention provides a polynucleotide encoding a bispecific antibody heavy chain in the form of an IgG-scFv that contains a full-length IgG and a scFv linked to the C-terminus and/or N-terminus of the full-length IgG. In another embodiment, the present invention provides a polynucleotide encoding a bispecific antibody light chain in the form of an IgG-scFv. In another embodiment, the present invention provides a recombinant vector comprising a polynucleotide encoding a heavy chain of a bispecific antibody, a polynucleotide encoding a light chain of a bispecific antibody, or both. According to another embodiment, the present invention relates to a recombinant cell transfected with a recombinant vector.

According to another embodiment, the present invention provides a method for producing a bispecific antibody, comprising expressing a polynucleotide encoding a heavy chain of the bispecific antibody, a polynucleotide encoding a light chain of the bispecific antibody, in a cell. The step of expressing the polynucleotide can be carried out by culturing a cell containing the polynucleotide (eg, in a recombinant vector) under conditions allowing expression of the polynucleotide. The method may further comprise isolating and/or purifying the bispecific antibody from the cell culture after the expression or culture step.

Brief description of drawings

In fig. 1A and 1B schematically illustrate an anti-PD-L1/anti-4-1BB bispecific antibody according to an embodiment.

In fig. 2 schematically illustrates the mechanism of action of an anti-PD-L1/anti-4-1BB bispecific antibody according to an embodiment.

In fig. 3 shows graphs demonstrating the selection criteria for PD-L1 variants in order to identify the necessary residues for Hu1210-41 binding.

In fig. 4 illustrates the locations of Y134, K162, and N183, residues (spheres) involved in binding to an anti-PD-L1 antibody according to an embodiment.

In fig. 5 shows that the anti-4-1BB antibodies of embodiments can bind to human 4-1BB with high affinity.

In fig. 6 shows that anti-4-1BB antibodies according to embodiments can effectively bind to 4-1BB expressed on mammalian cells.

In fig. 7A and 7B are graphs illustrating the binding of an anti-PD-L1/anti-4-1BB bispecific antibody according to an embodiment to human PD-L1 and human 4-1BB as measured by DACE (Dual Antigen Capture ELISA).

In fig. 8 shows that the anti-PD-L1/anti-4-1BB bispecific antibodies of the embodiment are stable in human serum.

In fig. 9A-9F show that the PD-L1x4-1BB bispecific antibodies of an embodiment activate 4-1BB signaling depending on the expression of PD-L1 on target cells.

In fig. 10A and 10B show the results of a PBMC assay for anti-PD-L1/anti-4-1BB bispecific antibodies according to embodiments. Also provided are graphs illustrating the T cell promoting activities of anti-PD-L1/anti-4-1BB bispecific antibodies according to embodiments.

In fig. 11 is a graph illustrating the tumor growth inhibition effect of the anti-PDL1/anti-4-1BB bispecific antibody according to the embodiment.

Detailed Disclosure of the Present Invention

Definitions.

It should be noted that the singular form of entity refers to one or more of these entities, for example, antibody should be understood as one or more antibodies. Thus, the singular form, the terms one or more, and at least one may be used interchangeably herein.

In the context of the present invention, the term polypeptide is intended to cover a single polypeptide as well as multiple polypeptides and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of product. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to designate a chain or chains of two or more amino acids are included in the definition of polypeptide, and the term polypeptide may be used instead of or interchangeably with any of these terms . The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be created by any method, including chemical synthesis.

The term isolated, as used herein in relation to cells, nucleic acids such as DNA or RNA, refers to molecules separated from other DNA or RNA, respectively, that are present in the natural source of the macromolecule. In the context of the present invention, the term isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when synthesized chemically. Moreover, the isolated nucleic acid is intended to include nucleic acid fragments that do not occur naturally as fragments and cannot be found in their natural state. In the context of the present invention, the term isolated also refers to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides.

In the context of the present invention, the term recombinant with respect to polypeptides or polynucleotides means a form of a polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be created by combining polynucleotides or polypeptides that do not normally occur together in nature.

Homology or identity or similarity refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the position in each of the sequences that may be aligned for comparison purposes. When a position in the sequence being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An unrelated or non-homologous sequence has less than 40% identity, although preferably less than 25% identity, with one of the sequences of the present invention.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (for example, 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99%) sequence identity with another sequence, meaning that when aligned this percentage of bases (or amino acids) is the same as when comparing two sequences. This alignment and the percentage of homology or sequence identity can be determined using software known in the art, for example, the software described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the default settings are used for alignment. One alignment software is BLAST using default parameters. Specifically, the software is BLASTN and BLASTP, which use the following default parameters: genetic code=standard, filter=none, strand=both, cutoff=60, wait=10, matrix=BLOSUM62, descriptions=50 sequences, sort by=HIGH ASSESSMENT, databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those that have the above percentage of homology and encode a polypeptide having the same or similar biological activity.

The term equivalent nucleic acid or polynucleotide refers to a nucleic acid having a nucleotide sequence having a certain degree of homology or sequence identity with the nucleic acid nucleotide sequence or its complement. A double-stranded nucleic acid homolog is intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology to the nucleic acid nucleotide sequence or its complement. In one aspect, nucleic acid homologues are capable of hybridizing with a nucleic acid or its complement. Likewise, an equivalent polypeptide refers to a polypeptide having a certain degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least at least 99%. In some aspects, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (eg, epitope binding) or structure (eg, salt bridge) of the reference sequence.

Hybridization reactions can be carried out under conditions of varying stringency. In general, the low stringency hybridization reaction is carried out at a temperature of approximately 40°C in approximately 10xSSC or a solution of equivalent ionic strength/temperature. Medium stringency hybridizations are typically performed at about 50°C in about 6xSSC, and high stringency hybridization reactions are typically performed at about 60°C in about 1xSSC. Hybridization reactions can also be carried out under physiological conditions, which are well known to one skilled in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH value and Mg ²⁺ concentration typically found in a cell.

- 6 045275

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) instead of thymine when the polynucleotide is RNA. Thus, the term polynucleotide sequence is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into databases on a central processing unit (CPU) computer and used for bioinformatics applications such as functional genomics and homology searching. The term polymorphism refers to the coexistence of more than one form of a gene or part of a gene. A part of a gene that has at least two different forms, i.e. two different nucleotide sequences is called a polymorphic region of a gene. A polymorphic region may be a single nucleotide whose identity differs between alleles.

The terms polynucleotide and oligonucleotide are used interchangeably and refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: gene or gene fragment (e.g., probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, miRNA, microRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be made before or after assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation to a labeling moiety. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form the double-stranded form.

The term encode, when used with respect to polynucleotides, refers to a polynucleotide that is said to encode a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA of the polypeptide and /or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from it.

In the context of the present invention, an antibody or antigen-binding polypeptide refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be the whole antibody and any of its antigen-binding fragments or one chain thereof. Thus, the term antibody includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having antigen binding biological activity. Examples thereof include, but are not limited to, a heavy or light chain complementarity determining region (CDR) or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region (FR) or any portion thereof, or at least one part of the binding protein.

In the context of the present invention, the terms antibody fragment or antigen binding fragment is a portion of an antibody such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv and the like. Regardless of structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term antibody fragment includes aptamers, spiegelmers and diabodies. The term antibody fragment also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

Single chain variable fragment or scFv refers to a fusion protein of the variable regions of the heavy (VH) and light chains ( _VL ) of immunoglobulins. In some aspects, the regions are connected by a short linker peptide consisting of 10-25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility, and can either connect the N-terminus of _VH to the C-terminus of _VL , or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of constant regions and the introduction of a linker. ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019.

The term antibody covers various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses among them (eg, γ1-γ4). It is the nature of the chain that determines the class of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, for example IgG1, IgG2, IgG3, IgG4, IgG5, etc.,

- 7 045275 are well characterized and are known to impart functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one skilled in the art given the present disclosure and are accordingly within the scope of the present disclosure. While all classes of immunoglobulins are clearly within the scope of the present disclosure, the following discussion will generally focus on the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Da and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a Y configuration, where the light chains fork over the heavy chains, starting at the Y mouth and continuing through the variable region.

Antibodies, antigen binding polypeptides, variants or derivatives thereof according to the present invention include, without limitation, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope binding fragments, for example, Fab, Fab' and F(ab') ₂ , Fd, Fvs, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing either the VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including eg anti-K antibodies to the LIGHT antibodies described herein). The immunoglobulin molecules or antibodies of the present disclosure can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

Light chains are classified as kappa or lambda (K, λ). Each heavy chain class can be coupled to either a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the tail portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated either by hybridomas or B cells or engineered host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the branched ends of the Y-configuration to the C-terminus at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used functionally. In this regard, it should be understood that the variable domains of both the light (VL) and heavy (VH) chain regions determine antigen recognition and specificity. In contrast, the light chain (CK) and heavy chain constant domains (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental motility, Fc receptor binding, complement fixation, and the like. Conventionally, the numbering of constant region domains increases as they become more distant from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region, the CH3 and CK domains actually containing the carboxy terminus of the heavy and light chain, respectively.

As stated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. Those. the VL domain and the VH domain or a subset of complementarity determining regions (CDRs) of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen binding site present at the end of each Y arm. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3). In some cases, such as certain immunoglobulin molecules derived from camelid species or derived from camelid immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains and no light chains. See, for example, Hamers-Casterman et al., Nature, 363:446-448 (1993).

In naturally occurring antibodies, the six complementarity determining regions or CDRs present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, called framework regions, exhibit less intermolecular variability. The framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that ensures the correct orientation of the CDRs through interchain non-covalent interactions. The antigen binding domain formed by the arranged CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids containing the CDRs and framework regions, respectively, for any given heavy or light chain variable region can be readily identified by one skilled in the art as they have been precisely defined (see www.bioinf.org.uk: Dr. Andrew C.R. Martin's Group; Sequences of Proteins of Immunological Interest, Kabat, E. et al., U.S. Department of Health and Human Services (1983); and Chothia and

Lesk, J. Mol. Biol., 196:901-917 (1987)).

Where there are two or more definitions of a term that are used and/or accepted in the art, the definition of the term used herein is intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term complementarity determining region (CDR) to describe the non-contiguous antigen binding sites of an antibody found in the variable region of both heavy and light chain polypeptides. This particular area has been described by Kabat et al., U.S., Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983); and Chothia et al., J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entirety. Kabat and Chotia's definitions of CDRs include overlaps or subsets of amino acid residues when compared to each other. However, it is intended that the use of any definition to refer to an antibody CDR or variants thereof is within the scope of the term as defined and used herein. The corresponding amino acid residues that include the CDRs as defined in each of the above references are presented in the table below for comparison. The exact residue numbers that span a particular CDR will vary depending on the sequence and the size of the CDR. Those skilled in the art can routinely determine which residues contain a particular CDR based on the amino acid sequence of the antibody variable region.

Kabat Table 1 Chotia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a variable domain sequence numbering system that applies to any antibody. One skilled in the art can clearly apply this Kabat numbering system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, Kabat numbering refers to the numbering system established in Kabat et al., U.S., Dept. of Health and Human Services, Sequence of Proteins of Immunological Interest (1983).

In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid position 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue . CDR-H2 begins at the fifteenth residue position after the end of CDR-H1, contains approximately 16-19 amino acids, and ends with the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue after the end of CDR-H2, spans 3-25 amino acids, and ends with the sequence WG-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (ie, after the cysteine residue), contains approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue position after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue position after the end of CDR-L2 (ie, after the cysteine residue), contains approximately 7-11 residues, and ends with the sequence F or W-G-X-G, where X is any amino acid.

The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, the variable region may be derived from cartilaginous fish (eg, sharks).

In the context of the present invention, the term heavy chain constant region includes amino acid sequences derived from the immunoglobulin heavy chain. A heavy chain constant region-containing polypeptide contains at least one of a CH1 domain, a hinge domain (eg, an upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use according to the present invention may comprise a polypeptide chain containing a CH1 domain, a polypeptide chain containing a CH1 domain, at least a portion of a hinge domain and a CH2 domain, a polypeptide chain containing a CH1 domain and a CH3 domain, a polypeptide chain containing a CH1, by

- 9 045275 at least part of a hinge domain and a CH3 domain, or a polypeptide chain containing a CH1 domain, at least part of a hinge domain, a CH2 domain and a CH3 domain. In another embodiment, the polypeptide of the present invention comprises a polypeptide chain containing a CH3 domain. In addition, an antibody for use according to the present invention may not contain at least a portion of the CH2 domain (eg, all or part of the CH2 domain). As set forth above, one skilled in the art will appreciate that the heavy chain constant region may be modified to differ in amino acid sequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody described herein may be derived from various immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term light chain constant region includes amino acid sequences derived from the light chain of an antibody. Preferably, the light chain constant region comprises at least one of a kappa constant domain or a lambda constant domain.

A light chain-heavy chain pair refers to a combination of a light chain and a heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As stated previously, the subunit structures and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. In the context of the present invention, the term VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term CH1 domain includes the first (most amino-terminal) domain of the immunoglobulin heavy chain constant region. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

In the context of the present invention, the term CH2 domain includes the portion of the heavy chain molecule that extends, for example, from approximately residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244-360, Kabat numbering system, and residues 231-340, EC system numbering; see Kabat et al., U.S., Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983). The CH2 domain is unique in that it is not paired with another domain. Rather, two N-linked branched carbohydrate the chains are located between the two CH2 domains of the intact native IgG molecule.It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and includes approximately 108 residues.

In the context of the present invention, the term hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region consists of approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be divided into three distinct domains: the upper, middle and lower hinge domains (Roux et al., J. Immunol., 161:4083 (1998)).

In the context of the present invention, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242, using the Kabat numbering system (position 226 or 229, EC numbering system).

In the context of the present invention, the term chimeric antibody means any antibody in which the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be intact, partial, or modified in accordance with the present invention) is derived from a second species. In some embodiments, the target region or binding site will be from a non-human source (eg, mouse or primate) and the constant region is human.

In the context of the present invention, the term percent humanization is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, and then dividing the resulting number by the total number of amino acids and multiplying by 100.

Specifically binds or has specificity in general means that an antibody binds to an epitope through its antigen binding domain, and that binding imparts some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to specifically bind to an epitope when it binds to that epitope through its antigen-binding domain more readily than it would bind to a random, unrelated epitope. In the context of the present invention, the term specificity is used to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be said to have higher specificity for a given epitope than antibody B, or antibody A may be said to bind to epitope C with higher specificity than to the related epitope D. Preferably, the antibody binds to the antigen (or epitope ) with high affinity, namely with a KD of 1x10 ^-7 M or less, more preferably 5x10 ^-8 M or less, more preferably 3x10 ^-8 M or less, more preferably 1x10 ^-8 M or less, more preferably 25x10 ^-9 M or less or even more preferably 1x10 ^-9 M or less.

In the context of the present invention, the terms treat or treatment can refer to both therapeutic treatment and prophylactic or prophylactic measures, where the goal is to prevent or slow (reduce) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization (i.e., no worsening) of disease status, delay or slowing of disease progression, alleviation or palliation of disease, and remission (partial or complete), whether detectable or undetectable. . Treatment may also mean an increase in life expectancy compared to life expectancy without treatment. Those in need of treatment include those who already have the condition or disorder, as well as those who are prone to acquiring the condition or disorder, or those whose condition or disorder needs to be prevented.

The term subject, or individual, or animal, or patient, or mammal can refer to any subject, particularly a mammal, for which diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, pets, farm animals, zoo animals, sporting or domestic animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

In the context of the present invention, phrases such as patient in need of treatment or subject in need of treatment include subjects, such as mammalian subjects, who will benefit from administration of an antibody or composition according to the present invention, used, for example, for detection, for diagnostic techniques and /or for treatment.

The present invention provides an anti-PD-L1/anti-4-1BB bispecific antibody capable of effectively blocking interactions between PD-L1 and its receptor PD-1 and between 4-1BB and its ligand. The bispecific antibody may have high binding affinity to both PD-L1 protein (eg, human PD-L1 protein) and 4-1BB protein (eg, human 4-1BB protein).

The anti-PD-L1/anti-4-1BB bispecific antibody may comprise an anti-PD-L1 antibody or an antigen-binding fragment thereof as a PD-L1-targeting moiety that is capable of specifically recognizing and/or binding to the PD-L1 protein, and anti -4-1BB antibody or antigen-binding fragment thereof as a 4-1BB-targeting moiety that is capable of specifically recognizing and/or binding to the 4-1BB protein.

Anti-PD-LI antibody.

The anti-PD-L1/anti-4-1BB bispecific antibody may contain an anti-PD-L1 antibody or an antigen binding fragment thereof as a PD-Ll targeting moiety. An anti-PD-LI antibody or antigen-binding fragment thereof may exhibit effective binding and inhibitory activities against PD-L1 and may be useful for therapeutic and diagnostic applications.

The PD-L1 protein is a type 1 transmembrane protein weighing 40 kDa. The PD-L1 protein may be a human PD-L1 protein, and the human PD-L1 protein may be selected without limitation from the group consisting of proteins listed in GenBank with accession number NP_001254635.1, NP_001300958.1, NP_054862.1, etc. d. Human PD-L1 protein includes an extracellular portion including the N-terminal immunoglobulin V (IgV) domain (amino acids 19-127) and the C-terminal immunoglobulin C (IgC) domain (amino acids 133-225). Unlike pre-existing anti-PD-L1 antibodies, which bind to the IgV domain of PD-L1, thereby disrupting the binding between PD-1 and PD-L1, the anti-PD-L1 antibody or fragment thereof contained in the bispecific antibody cannot bind to the immunoglobulin V (IgV) domain of PD-L1 protein, but binds to the IgC domain of PD-L1 to effectively inhibit PD-L1, thereby improving therapeutic effects.

In particular, an anti-PD-L1 antibody or fragment thereof contained in a bispecific antibody can specifically bind to the immunoglobulin C (IgC) domain of the PD-L1 protein. In the case of human PD-L1 protein, the IgC domain contains or consists essentially of amino acid residues 133-225 of the full length human PD-L1 protein. More specifically, the anti-PD-L1 antibody or fragment thereof can bind to at least one residue selected from amino acid residues Y134, K162 and N183 of the human PD-L1 protein. In some embodiments, an anti-PD-L1 antibody or fragment thereof can bind to at least two residues selected from amino acid residues Y134, K162, and N183 of the human PD-L1 protein. In some embodiments, the anti-PD-L1 antibody or fragment thereof is incapable of binding to the immunoglobulin V (IgV) domain of the PD-L1 protein, wherein the IgV domain consists of amino acid residues 19-127 of the human PD-L1 protein.

In an embodiment, the anti-PD-L1 antibody or fragment thereof is capable of exhibiting specificity for human PD-L1 protein.

The anti-PD-LI antibody or fragment thereof may comprise (1) a CDR1 VH having the amino acid sequence of SEQ ID NO: 1;

(2) a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3;

(3) a CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 262, 263, 264, 265, 266 and 267;

(4) CDR1 VL having the amino acid sequence of SEQ ID NO: 6, 268 and 269;

(5) CDR2 VL having the amino acid sequence of SEQ ID NO: 7; and (6) CDR3 VL having the amino acid sequence of SEQ ID NOs: 8, 270, 271 and 272.

table 2

Name CDR of anti-PD-L1 antibodies Sequence SEQ ID NO: CDR1VH SYDMS 1 CDR2 VH TIS D AGG YIY YS DS V KG 2 TISDAGGYIYYRDSVKG 3 CDR3VH EFGKRYAEDY 4 ELPWRYALDY 5 EFGKRYALDS 262 EIFNRYALDY 263 ELHFRYALDY 264 ELYFRYALDY 265 ELLHRYALDY 266 ELRGRYALDY 267 CDR1VL KASQDVTPAVA 6 KAKQDVTPAVA 268 KASQDVWPAVA 269 CDR2 VL STSSRYT 7 CDR3 VL QQHYTTPLT 8 MQHYTTPLT 270 QQHSTTPLT 271 QQHSDAPLT 272

In some embodiments, the antibody or fragment thereof includes no more than one, no more than two, or no more than three of the above substitutions. In some embodiments, the antibody or fragment thereof comprises CDR1 VH sequences SEQ ID NO: 1, CDR2 VH sequences SEQ ID NO: 2 or 3, CDR3 VH sequences SEQ ID NO: 4, 5, 262, 263,264, 265,266 or 267, CDR1 VL sequences SEQ ID NO: 6, 268 or 269, CDR2 VL sequences SEQ ID NO: 7 and CDR3 VL sequences SEQ ID NO: 8, 270, 271 or 272.

For example, an anti-PD-L1 antibody or fragment thereof may comprise a CDR1 VH having the amino acid sequence of SEQ ID NO: 1, a CDR2 VH having the amino acid sequence of SEQ ID NO: 2 or 3, a CDR3 VH having the amino acid sequence of SEQ ID NO: 4 or 5, CDR1 VL having the amino acid sequence of SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8.

Back mutations may be useful in preserving certain characteristics of anti-PDL1 antibodies. In some embodiments, the anti-PD-L1 antibodies of the present invention, particularly human or humanized antibodies, may include one or more back mutations. In some embodiments, the back mutation (i.e., inserting an amino acid at a specific position) in the heavy chain variable region (VH) is one or more selected from (a) Ser at position 44, (b) Ala at position 49, ( c) Ala at position 53, (d) Ile at position 91, (e) Glu at position 1, (f) Val at position 37, (g) Thr at position 40, (h) Val at position 53, (i) Glu at position 54, (j) Asn at position 77, (k) Arg at position 94, and (l) Thr at position 108 of the heavy chain variable region, according to Kabat numbering, and combinations thereof. In some embodiments, the back mutations in VH are selected from (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d) Ile at position 91 of the heavy chain variable region as numbered by Kabatu and their combinations.

In some embodiments, the back mutation in the light chain variable region (VL) is one or more selected from (a) Ser at position 22, (b) Gln at position 42, (c) Ser at position 43, (d) Asp at position 60 and (e) Thr at position 63 of the light chain variable region according to Kabat numbering and combinations thereof.

In some embodiments, the anti-PD-L1 antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or combinations thereof. In some embodiments, the light chain constant region may be a kappa or lambda chain constant region. In some embodiments, the antibody is of an IgG, IgM, IgA, IgE, or IgD isotype, such as human IgG, human IgM, human IgA, human IgE, or human IgD. In some embodiments, the isotype may be IgG, for example, human IgG such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the fragment (an anti-PD-L1 antibody antigen-binding fragment) may be any fragment comprising a heavy chain CDR and/or a light chain CDR of an antibody, and, for example, may be selected from the group consisting of Fab, Fab', F(ab')2, Fd (containing a heavy chain variable region and a CH1 domain), Fv (a heavy chain variable region and/or a light chain variable region), single chain Fv (scFv containing or consisting essentially of a heavy chain variable region and light chain variable region, in any order, and a peptide linker between the heavy chain variable region and the light chain variable region), single chain antibodies, disulfide-linked Fv (sdFv), and the like.

An anti-PD-LI antibody or fragment thereof is, without limitation, a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or fragment thereof is not naturally occurring or is chemically or recombinantly synthesized.

The binding of an antibody of the present invention to PD-L1 can be assessed using one or more methods well known in the art. For example, in a preferred embodiment, the antibody can be tested using a flow cytometry assay in which the antibody reacts with a cell line expressing human PD-L1, such as CHO cells, that have been transfected to express PD-L1, such as human PD-L1 or simian PD-L1, for example, rhesus or cynomolgus monkeys, or murine PD-L1, on their cell surface. Other suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4+ activated T cells that express native PD-L1. Other suitable binding assays include ELISA assays, for example using recombinant PD-L1 protein. Additionally or alternatively, antibody binding, including binding kinetics (eg, KD value), can be tested in the Biacore assay. Preferred binding affinities of the antibodies of the present invention include those with a dissociation constant or KD of 4.25x10'9 M or less.

Given that each of these antibodies can bind to PD-L1, such as human PD-L1, the CDR sequences or V _H and VL sequences can be mixed and coincident with the creation of other anti-PD-L1 binding molecules according to the present invention. Preferably, the CDR sequences or chains V _H and VL are mixed or matched, for example, the V _H sequence from a particular V _H /V _L pair is replaced by a structurally similar VH sequence. Likewise, preferably the VL sequence from a particular VH/VL pair is replaced by a structurally similar VL sequence.

Anti-4-1BB antibody.

The anti-PD-L1/anti-4-1BB bispecific antibody may contain an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB-targeting moiety.

In an embodiment, the anti-4-1BB antibody or fragment thereof may specifically bind to a 4-1BB protein (eg, human 4-1BB).

- 13 045275

For example, the human 4-1BB protein may be selected without limitation from the group consisting of proteins submitted to NCBI under accession number NP_001552, etc. These anti-4-1BB antibodies or antigen binding fragments thereof are capable of enhancing the immune response and/or treating a tumor (cancer) in a mammal. An anti-4-1BB antibody or antigen-binding fragment thereof is characterized by localization and/or activation only in the tumor microenvironment (TME) and/or significantly reduced liver toxicity compared to pre-existing anti-4-1BB antibodies, while maintaining the effectiveness of enhancing the immune response and /or tumor treatment.

The term 4-1BB refers to CD137 or TNFRSF9 (TNF receptor superfamily 25 member 9), is a member of the TNF receptor superfamily (TNFRSF) and is a costimulatory molecule that is expressed upon activation of immune cells, both innate immune cells and adaptive immune cells. systems. In the context of the present invention, 4-1BB may be derived from mammals, for example Homo sapiens (human) (NCBI accession number NP_001552). For example, human protein 4-1BB (NP_001552) can be represented by the amino acid sequence (SEQ ID NO: 9) as shown below:

mgnscyniva tlllvlnfcr trslqdpcsn cpagtfcdnn rnqicspcpp nsfssaggqr

161 tcdicrqckg vfrtrkecss tsnaecdctp gfhclgagcs mceqdckqgq eltkkgckdc

121 cfgtfndqkr gicrpwtncs Idgksvlvng tkerdvvcgp spadlspgas svtppapare

181 pghspqiisf flaltstall fllffltlrf svvkrgrkkl lyifkqpfmr pvqttqeedg

241 cscrfpeeee ggcel

As described herein, the term 4-1BB includes variants, isoforms, homologs, orthologs and paralogs. For example, antibodies specific for the human 4-1BB protein may, in some cases, cross-react with the 4-1BB protein of other non-human species. In other embodiments, antibodies specific for human 4-1BB protein may be completely specific for human 4-1BB protein and may exhibit species or other types of cross-reactivity or may cross-react with 4-1BB of other specified species, but not all other species (eg, cross-reacts with monkey 4-1BB but not with murine 4-1BB). The term human 4-1BB refers to human 4-1BB sequence, such as the complete amino acid sequence of human 4-1BB having NCBI accession number NP_001552. The term murine 4-1BB refers to a murine 4-1BB sequence, such as the complete amino acid sequence of murine 4-1BB having NCBI accession number NP 033430.1. 4-1BB may also be known in the art, for example CD137. The sequence of human 4-1BB according to the present disclosure may differ from the sequence of human 4-1BB with NCBI accession number NP_001552, for example, by the presence of conserved mutations or mutations in non-conserved regions, and 4-1BB according to the present disclosure has substantially the same biological function as human 4-1BB with NCBI registration number NP_001552.

As shown in the experimental examples, the anti-4-1BB antibodies disclosed herein exhibit abilities to bind to 4-1BB, abilities to bind to 4-1BB that is expressed on the cell surface, and high binding affinities to 4-1BB. In addition, as demonstrated in the Experimental Example, the anti-4-1BB antibody disclosed herein, especially in combination with the α-anti-PD-L1 antibody disclosed herein, is capable of activating T cells. In addition, as shown in the experimental example, the anti-4-1BB antibody disclosed herein has an enhanced antitumor effect in vivo.

These anti-4-1BB antibodies may be useful for therapeutic purposes such as treatment of various types of cancer, etc., and may also be useful for diagnostic and prognostic purposes.

In an embodiment, the anti-4-1BB antibody or fragment thereof is capable of specificity for human 4-1BB protein. The anti-4-1BB antibody or fragment thereof may comprise (i) a CDR1 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11;

(ii) a CDR2 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13;

(iii) a CDR3 VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16 and 17;

(iv) CDR1 VL having the amino acid sequence of SEQ ID NO: 18;

(v) CDR2 VL having the amino acid sequence of SEQ ID NO: 19; and (vi) CDR3 VL having the amino acid sequence of SEQ ID NO: 20.

For example, an anti-4-1BB antibody or fragment thereof may comprise a CDR1 VH having the amino acid sequence of SEQ ID NO: 10 or 11, a CDR2 VH having the amino acid sequence of SEQ ID NO: 12 or 13, a CDR3 VH having the amino acid sequence of SEQ ID NO: : 14, 15, 16 or 17, CDR1 VL, having the amino acid sequence SEQ ID NO: 18, CDR2 VL,

- 14 045275 having the amino acid sequence SEQ ID NO: 19, and CDR3 VL having the amino acid sequence SEQ ID NO: 20.

Table 3

Name CDR1VH CDR of anti-4-1BB antibodies Sequence SEQ ID NO: SYDMS 10 GYDMS eleven CDR2 VH WISYSGGSIYYADSVKG VIYPDDGNTYYADSVKG 12 13 CDR3 VH DGQRNSMREFDY HGGQKPTTTKSSSAYGMDG DAQRNSMREFDY DAQRQSMREFDY 14 15 16 17 CDR1VL SGSSSNIGNNYVT 18 CDR2 VL ADSHRPS 19 CDR3 VL ATWDYSLSGYV 20

In non-limiting examples of an anti-4-1BB antibody or fragment thereof, (1) the heavy chain variable region may comprise or consist essentially of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23 and 24 , or a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity, or at least 99% with the amino acid sequences described above, and/or (2) the light chain variable region may contain or consist essentially of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 26, or a polypeptide having sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the amino acid sequences described above.

Non-limiting examples of an anti-4-1BB antibody or fragment thereof may comprise a heavy chain variable region containing or essentially consisting of the amino acid sequence of SEQ ID NO: 21, 22, 23 or 24.

EVQLLESGGGLVQPGGSLRLSCAASGFFSSYDMSWVRQAPGKCLEWVSWISYS

GGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGQRNSMREFDYW

GQGTLVTVSS (SEQ ID NO: 21)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVIYPD DGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDAAVYYCAKHGGQKPTTKSSSAY GMDGWGQGTLVTVSS (SEQ ID NO: 22)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYS GGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYW GQGTLVTVSS (SEQ ID NO: 23)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYS GGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRQSMREFDYW GQGTLVTVSS (SEQ ID NO: 24)

Non-limiting examples of an anti-4-1BB antibody or fragment thereof may comprise a light chain variable region containing or substantially consisting of the amino acid sequence of SEQ ID NO: 25 or 26.

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QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRP SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL (SEQ ID NO: 25)

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRP

SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL (SEQ ID

NO: 26)

The antibodies of the present invention are characterized by specific functional characteristics or properties of the antibodies. For example, antibodies specifically bind to human 4-1BB and may bind to 4-1BB of some other species, e.g., simian 4-1BB, e.g., cynomolgus monkeys, rhesus macaques, but may not bind substantially to 4-1BB of some other species , for example, mouse 4-1BB. Preferably, the antibody of the present invention binds to human 4-1BB with high affinity.

In addition, the antibody of the present invention, particularly as a bispecific antibody containing the anti-PD-L1 antibody of the present invention, has the ability to enhance immune cell proliferation, survival, cytokine secretion and cytolytic activity of CD8 T cells. In certain embodiments, an antibody of the present invention, particularly as a bispecific antibody comprising an anti-PD-L1 antibody of the present invention, binds to human 4-1BB and exhibits the ability to activate T cells.

Other means of assessing the ability of an antibody to stimulate an immune response include the ability of the antibody to inhibit tumor growth, for example, in an in vivo tumor graft model.

The binding of the antibody of the present invention to 4-1BB can be assessed using one or more methods well known in the art. For example, in a preferred embodiment, the antibody can be tested using a flow cytometry assay in which the antibody reacts with a cell line expressing human 4-1BB, such as CHO cells, that have been transfected to express 4-1BB, such as human 4-1BB or simian 4-1BB, for example rhesus or cynomolgus monkeys, or murine 4-1BB, on their cell surface. Other suitable cells for use in flow cytometry assays include anti-CD3-stimulated CD4+ activated T cells that express native 4-1BB. Other suitable binding assays include ELISA assays, for example using recombinant 4-1BB protein.

Additionally or alternatively, antibody binding, including binding kinetics (eg, KD value), can be tested in the Octet assay. Preferred binding affinities of the antibodies of the present invention include those with a dissociation constant or KD of 1.80 x 10 ^-9 M or less.

In some embodiments, the anti-4-1BB antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or combinations thereof. In some embodiments, the light chain constant region may be a kappa or lambda chain constant region. In some embodiments, the antibody is of an IgG, IgM, IgA, IgE, or IgD isotype, such as human IgG, human IgM, human IgA, human IgE, or human IgD. In some embodiments, the isotype may be IgG, such as human IgG such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the fragment (an anti-PD-L1 antibody antigen-binding fragment) may be any fragment comprising a heavy chain CDR and/or a light chain CDR of an antibody, and, for example, may be selected from the group consisting of Fab, Fab', F(ab') ₂ , Fd (containing a heavy chain variable region and a CH1 domain), Fv (a heavy chain variable region and/or a light chain variable region), single chain Fv (scFv containing or essentially consisting of a heavy chain variable region and light chain variable region, in any order, and a peptide linker between the heavy chain variable region and the light chain variable region), single chain antibodies, disulfide-linked Fv (sdFv), and the like.

An anti-4-1BB antibody or fragment thereof is, without limitation, a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or fragment thereof is not naturally occurring or is chemically or recombinantly synthesized.

Given that each of these antibodies can bind to 4-1BB, such as human 4-1BB, the CDR sequences or VH and _VL sequences can be mixed and coincident with the creation of other anti-4-1BB binding molecules according to the present invention. Preferably, the VH and VL CDR sequences or chains are mixed or matched, eg, a VH sequence from a particular VH/VL pair is replaced by a structurally similar VH sequence. Likewise, it is preferable to have a VL sequence from a particular pair

- 16 045275

VH/VL is replaced by a sequence similar in structure to V _L .

Anti-PD-L1/anti-4-1BB bispecific antibody.

In a bispecific antibody comprising a PD-L1-targeting moiety and a 4-1BB-targeting moiety, one of the PD-L1-targeting moiety and the 4-1BB-targeting moiety may be a full-length antibody and the other may be an antigen binding moiety (eg, scFv ), containing a heavy chain CDR, a light chain CDR, or a combination thereof. A full-length antibody targeting one of the PD-L1 and 4-1BB proteins and an antigen binding fragment targeting the other protein may be chemically linked (eg, covalently linked) directly or through a peptide linker. The antigen-binding fragment (eg, scFv) can be linked directly or via a peptide linker to the N-terminus of a full-length antibody (eg, the N-terminus of the light chain or heavy chain of a full-length antibody), the C-terminus of a full-length antibody (eg, the C-terminus of heavy chain (or Fc or CH3 domain) of a full-length antibody) or both (see Fig. 1A and 1B).

In an embodiment, the bispecific antibody may comprise a full-length anti-PD-L1 antibody, an antigen binding fragment (eg, scFv) of an anti-4-1BB antibody, and a peptide linker therebetween. In another embodiment, the bispecific antibody may comprise a full-length anti-4-1BB antibody, an antigen binding fragment (eg, scFv) of an anti-PD-L1 antibody, and a peptide linker therebetween.

In an embodiment, the scFv contained in the bispecific antibody may comprise a heavy chain variable region and a light chain variable region in any order. For example, the scFv contained in a bispecific antibody may comprise a heavy chain variable region and a light chain variable region in an N-terminal to C-terminal direction, and optionally a peptide linker therebetween, or alternatively, the scFv contained in a bispecific antibody may contain a light chain variable region. chains and a heavy chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween.

In an embodiment, the anti-PD-L1/anti-4-1BB bispecific antibody activates 4-1BB signaling and the resulting immune response depending on the PD-L1 expressed on cell surfaces.

The use of a peptide linker for a bispecific antibody can result in high purity of the antibody.

In the context of the present invention, the term peptide linker can include any amino acids from 1 to 100, in particular from 2 to 50, and any kind of amino acids can be included without any limitation. The peptide linker may include, for example, Gly, Asn and/or Ser residues, and may also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker may be known in the relevant field. Meanwhile, the length of the peptide linker can be determined differently within a limit such that the functions of the fusion protein will not be affected. For example, a peptide linker may be formed by including a total of from about 1 to about 100, from about 2 to about 50, or from about 5 to about 25 of one or more amino acids selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker can be represented by (GmS1) _n (m, 1, and n are independently an integer from about 1 to about 10, particularly an integer from about 2 to about 5). For example, examples of peptide linkers are summarized as follows:

- 17 045275

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In another embodiment, both the PD-L1 targeting moiety and the 4-1BB targeting moiety may be a full length antibody or antigen binding fragment comprising a heavy chain CDR, a light chain CDR, or a combination thereof.

In another embodiment, the bispecific antibody may be in a heterodimeric form that comprises a first arm including a pair of a first heavy chain and a first light chain targeting one of PD-L1 and 4-1BB, and a second arm including a pair of a second heavy chain and a second light chain targeting the other.

In an embodiment, the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, such as human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen binding fragment may be selected from the group consisting of Fab, Fab', F(ab') ₂ , Fd, Fv, scFv, single chain antibodies, sdFv and the like, as described above. For example, the full-length antibody may be in the form of a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4) and the antigen binding fragment may be a scFv.

For example, an antibody described herein may contain a flexible linker sequence or may be modified to add a functional moiety (eg, PEG, drug, toxin, or tag).

Antibodies or variants described herein may contain derivatives that are modified, for example, by covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to an antigen (eg, epitope). For example, without limitation, antibodies can be modified, for example, by at least one selected from the group consisting of glycosylation, acetylation, pegylation, phosphorylation

- 18 045275 phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known methods, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, antibodies may contain one or more non-classical amino acids.

Antibodies or fragments thereof can be labeled with a detectable label by labeling (binding) with conventional labeling agents selected from chemiluminescent compounds, fluorescent compounds (eg, fluorescent emitting metals), radioisotopes, dyes, etc. The presence of labeled antibodies or fragments thereof can be detected by measuring the signal generated by the chemical reaction between the antibody (or fragment thereof) and the labeling agent. Examples of a particularly suitable labeling agent may be at least one selected from the group consisting of luminol, isoluminol, acridinium theromatic ester, imidazole, acridinium salt, oxalate ester, fluorescent emitting metals, and the like. For example, the metals emitting fluorescence may be ¹⁵² Eu or other lanthanides. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In certain embodiments, the resulting bispecific antibodies will not induce a harmful immune response in the animal being treated, such as a human. In one embodiment, the bispecific antibody can be modified to reduce its immunogenicity using any conventional techniques. For example, a bispecific antibody may be a humanized, primatized, deimmunized, or chimeric antibody. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This can be achieved in a variety of ways, including (a) transplanting entire non-human variable domains onto human constant regions to form chimeric antibodies;

(b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) into a human framework and constant regions, with or without retention of critical residues of the framework; or (c) transplanting entire non-human variable domains but masking them with a human-like region by replacing surface residues.

Deimmunization can also be used to reduce the immunogenicity of the antibody. In the context of the present invention, the term deimmunization may include alteration of an antibody with modification of T cell epitopes (see, for example, International Application Publications No. WO/9852976 A1 and WO/0034317 A2). For example, the heavy chain variable sequences and light chain variable sequences of the parent antibody are analyzed and a map of human T cell epitopes from each V region is generated showing the location of the epitopes in relation to complementarity determining regions (CDRs) and other key residues in the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A series of alternative heavy chain variable sequences and light chain variable sequences are developed, including combinations of amino acid substitutions, and these sequences are then included in a series of binding polypeptides. Typically, 12 to 24 variant antibodies are generated and tested for binding and/or function. The complete heavy and light chain genes containing the modified variable and human constant regions are then cloned into expression vectors and subsequent plasmids introduced into cell lines to produce the complete antibody. The antibodies are then compared in appropriate biochemical and biological assays to determine the best option.

The binding specificity and/or affinity of the bispecific antibody to each protein target can be determined using any conventional assay, for example, but not limited to, in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Alternatively, the methods described for producing single chain units (US Pat. No. 4,694,778, etc.) can be adapted to produce single chain units according to the present invention. Single-chain units are formed by linking heavy and light chain fragments of the Fv region through an amino acid bridge (peptide linker), resulting in a single-chain fusion peptide (scFv). Methods for assembling functional Fv fragments in E. coli can also be used.

Examples of techniques that can be used to produce single chain Fv (scFv) and anti

- 19 045275 bodies, include methods described in US patents No. 4946778, 5258498, etc.). For some applications, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferable. A chimeric antibody is a molecule in which different parts of the antibody come from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, for example, US Pat. Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

Humanized antibodies are antibody molecules derived from antibodies from a non-human species that bind a desired antigen, having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in human framework regions are replaced with a corresponding residue from a CDR donor antibody to alter, preferably improve, antigen binding. These scaffold substitutions are identified by methods well known in the art, for example, by modeling interactions of CDRs and scaffold residues to identify scaffold residues important for antigen binding, and sequence comparisons to identify unusual scaffold residues at specific positions (see, e.g., Queen et al., US Pat. No. 5,585,089, which are incorporated herein by reference in their entirety). Antibodies can be humanized using various methods known in the art, including, for example, CDR grafting (US Pat. No. 5,225,539, 5,530,101, 5,585,089, etc., each of which is incorporated by reference in its entirety), masking of surface residues or repositioning (EP 592106; EP 519596, each of which is incorporated by reference in its entirety) and circuit reversal (US Pat. No. 5,565,332, which is incorporated by reference in its entirety).

Fully human antibodies are particularly preferred for the therapeutic treatment of human patients. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also US patents. No. 4444887, 4716111, etc., each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable region, constant region and diversity region can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes.

The mouse immunoglobulin heavy and light chain genes can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, for example all or part of the desired target polypeptide. Monoclonal antibodies directed against an antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutation. Thus, using this method, therapeutically useful IgG, IgA, IgM and IgE antibodies can be obtained.

Fully human antibodies that recognize a selected epitope can also be produced using a method called controlled selection. In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to guide the selection of a fully human antibody recognizing the same epitope.

In another embodiment, DNA encoding the desired monoclonal antibodies can be readily isolated and sequenced using conventional techniques (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells serve as the preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins . More specifically, the isolated DNA (which may be synthetic, as described herein) can be used to clone constant and variable region sequences to obtain

- 20045275 antibodies, as described in Newman et al., US patent No. 5658570, which is incorporated herein by reference. Essentially, this results in RNA being extracted from selected cells, converted into cDNA, and amplified by PCR using Ig-specific primers. Suitable primers for this purpose are also described in US Pat. No. 5,658,570. As will be described in more detail below, transformed cells expressing the desired antibody can be grown in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.

In addition, through the use of conventional recombinant DNA techniques, one or more CDRs of a bispecific antibody can be inserted into framework regions, such as human framework regions, in order to humanize the non-human antibody. The framework regions can be naturally occurring or consensus framework regions, and preferably human framework regions (see, for example, Chothia et al., J. Mol. Biol., 278:457-479 (1998) for a list of human framework regions). For example, a polynucleotide generated by the combination of framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, for example, LIGHT. Preferably, one or more amino acid substitutions may be made in the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen (or epitope). In addition, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues involved in an intrachain disulfide bond to create antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotide are covered by the present disclosure and are within the skill of the person skilled in the art.

In addition, techniques developed to produce chimeric antibodies by splicing genes from a mouse antibody molecule with the corresponding antigen specificity together with genes from a human antibody molecule with the corresponding biological activity can be used. In the context of the present invention, a chimeric antibody is a molecule in which different parts are derived from different animal species, for example, having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to one of ordinary skill in the art. Such techniques are described in various laboratory manuals and primary publications.

In addition, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding an antibody of the present invention, including, without limitation, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, using saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

Non-limiting examples of anti-PD-L1/anti-4-1BB bispecific antibodies are shown in table. 4 below.

In the context of the present invention, the heavy component means the anti-PD-L1/anti-41BB component of the bispecific antibody according to the present invention, which contains (1) a heavy chain of an anti-PD-L1 antibody and (2) a heavy chain and a light chain of anti-4-1BB antibodies.

In the context of the present invention, the light component means the anti-PD-L1/anti-41BB component of the bispecific antibody according to the present invention, which contains the light chain of the anti-PD-L1 antibody.

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Table 4

Examples of anti-PD-L1/anti-4-1BB bispecific antibody

ABLPNB.01 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYSDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICAREFG KRYALDYWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS SEQ ID NO: 27

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASG TPGRRVTISCSGSSSNIGNNY VTWYQQLPGTAPK LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSE DEADYYCATWDYSLSGYVFGCGTKLTVLGGGGS GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWI SYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARDG QRNSMREFDYWGQGTLV TVSS Easy component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPRE AKVQWKVDNALQS GNS QES VTEQDS KDST YSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 28 ABLPNB.02 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYSDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICAREFG KRYALDYWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASG TPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPK SEQ ID NO: 29

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LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSE DEADYYCATWDYSLSGYVFGCGTKLTVLGGGGS GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWI SYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARDAQRNSMREFDYWGQ GTLV TVSS Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPRE AKVQWKVDNALQS GNS QES VTEQDS KDST YSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: thirty ABLPNB.03 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYSDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICAREFG KRYALDYWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYASTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSASG TPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPK LLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSE DEADYYCATWDYSLSGYVFGCGTKLTVLGGGG S GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSGYDMSWVRQAPGKCLEWVSVI YPDDGNTYYADSVKGRFTISRDNSKNTLYLQMN SEQ ID NO: 31

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SLRAEDAAVYYCAKHGGQKPTTTKSSSAYGMDG WGQGTLVTVSS Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ GO NO: 32 ABLPNB.04 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVLGG G GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVS WISYSGGSIYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDGQRNSMREFDYWGQGT LVTVSS SEQ GO NO: 33

- 24 045275

Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 34 ABLPNB.05 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTS GGT AALGCLVKD YFPEPVTVS WNS GALTS G VHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTK LVTVSS SEQ GO NO: 35 Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST SEQ ID NO: 36

- 25 045275

YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC ABLPNB.06 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVLGG G GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFFSSYDMSWVRQAPGKCLEWVS WISYSGGSIYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDAQRQSMREFDYWGQGT LVTVSS SEQ ID NO: 37 Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 38

- 26 045275

ABLPNB.07 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVL GGG GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFFSSYDMSWVRQAPGKCLEWVS WISYSGGSIYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDAQRNSMREFDYWGQGT LVTVSS SEQ ID NO: 39 Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 40

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ABLPNB.O8 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVL GGG GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFFSSYDMSWVRQAPGKCLEWVS WISYSGGSIYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARDAQRQSMREFDYWGQGT LVTVSS SEQ ID NO: 41 Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 42

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ABLPNB.09 Heavy component EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDM SWVRQAPGKSLEWVATISDAGGYIYYRDSVKGR FTISRDNAKNSLYLQMNSLRDEDTAVYICARELP WRYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSQSVLTQPPSAS GTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAP KLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRS EDEADYYCATWDYSLSGYVFGCGTKLTVLGG G GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFSGYDMSWVRQAPGKCLEWVS VIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDAAVYYCAKHGGQKPTTKSSSAYGM DGWGQGTLVTVSS SEQ ID NO: 43 Light component DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVA WYQQKPGKAPKLLIYSTSSRYTGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO: 44

Therapeutic use of bispecific antibodies.

The bispecific antibody of the present invention is capable of simultaneously blocking the activities of PD-L1 and 4-1BB, thereby exhibiting improved effects in immunotherapy and/or cancer treatment, for example, by activating an immune response (see FIG. 2). Given the ability of the bispecific antibodies of the present invention to inhibit the binding of PD-L1 to PD-1 molecules and stimulate antigen-specific T cell responses, the present invention also provides compositions or in vitro and in vivo methods for using, enhancing or up-regulating the antibodies of the present invention antigen-specific T-cell responses.

According to an embodiment, the present invention relates to a pharmaceutical composition containing a bispecific antibody as described above. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to stimulate an immune response (eg, an antigen-specific T-cell response) and/or treat and/or prevent a disease associated with PD-L1, 4-1BB, or both.

In another embodiment, the present invention provides a method of stimulating an immune response (eg, an antigen-specific T cell response) and/or treating and/or preventing a disease associated with PD-L1, 4-1BB, or both, in subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the bispecific antibody or pharmaceutical composition. The method may further include identifying a subject in need of treatment and/or prevention of a disease associated with PD-L1, 4-1BB, or both, prior to the administration step.

The disease associated with PD-L1, 4-1BB, or both may be selected from cancer (or tumors), infectious diseases, autoimmune reactions, nervous system disorders, and the like.

In an embodiment, the subject may be selected from mammals, including humans, for example, a mammal (eg, human) having mammalian cancer cells. In another embodiment, the subject may be a cell separated from a mammal, such as a mammal suffering from a disease selected from

- 29 045275 cancer, infectious diseases, autoimmune reactions, nervous system disorders, etc. (eg, a cancer cell or a cell isolated from an infectious site in a mammal, or a T cell such as a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof).

According to another embodiment, the present invention relates to the use of a bispecific antibody or pharmaceutical composition for the treatment and/or prevention of cancer. According to another embodiment, the present invention relates to the use of a bispecific antibody for the preparation of a pharmaceutical composition for the treatment and/or prevention of cancer.

In the pharmaceutical compositions, methods and/or uses of the present invention, disease associated with PD-L1, 4-1BB or both may be associated with activation (eg, abnormal activation or over-activation) and/or excess production of ( overexpression) of PD-L1, 4-1BB, or both. For example, the disease could be cancer.

The cancer may be a solid cancer or a blood cancer, preferably a solid cancer.

Administration of the bispecific antibody can be accomplished by one or more methods well known in the art.

A therapeutically effective dose of an antibody of the present invention preferably results in a reduction in the severity of symptoms of a disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of deterioration or disability due to the disease. For example, for treating tumor-bearing subjects, a therapeutically effective dose preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even more preferably by at least approximately 80% compared to untreated subjects. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject, which is typically a human or other mammal.

The pharmaceutical compositions may contain an effective amount of the bispecific antibody and an acceptable carrier. In some embodiments, the composition further includes a second antitumor agent (eg, an immune checkpoint inhibitor).

In a specific embodiment, the term pharmaceutically acceptable may refer to approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more particularly, in humans. In addition, the pharmaceutically acceptable carrier is generally a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material or excipient for preparing any type of formulation.

A composition containing an antibody or antigen-binding fragment thereof according to the present invention may further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is one commonly used in the preparation of the composition.

Diagnostic use of bispecific antibodies.

Overexpression and/or excessive activation of PD-L1 and/or 4-1BB is observed in a biological sample (eg, cells, tissue, blood, serum, etc.) taken from a patient suffering from a certain cancer (eg, tumor cell), and/or patients who have cells overexpressing PD-L1 and/or 4-1BB are likely to respond to bispecific antibody treatment. Accordingly, the bispecific antibody of the present invention can also be used for diagnostic and prognostic purposes.

In an embodiment, the present invention provides a pharmaceutical composition for diagnosing a disease associated with PD-L1, 4-1BB, or both, the composition comprising a bispecific antibody. In another embodiment, the present invention relates to the use of a bispecific antibody for diagnosing a disease associated with PD-L1, 4-1BB, or both.

In another embodiment, the present invention provides a method for diagnosing a disease associated with PD-L1, 4-1BB, or both, the method comprising contacting a biological sample taken from a patient with a bispecific antibody and detecting an antigen-antigen response. antibody or measurement of the level of antigen-antibody reaction in a biological sample. According to this method, when an antigen-antibody reaction is detected in a biological sample or the level of an antigen-antibody reaction in a biological sample is higher than that in a normal sample, the patient from whom the biological sample is collected can be determined as a patient suffering from a PD-related disease. L1, 4-1BB, or both. Therefore, in some embodiments, the method may further comprise contacting a normal sample with a bispecific antibody and measuring the level of antigen-antibody reaction in the normal sample. In addition, the method may further include comparing the level of antigen-antibody reaction in the biological sample and in the normal sample after the measurement step. In addition, after the detection step or comparison step, the method may further include determining the patient as

- 30 045275 a patient with a disease associated with PD-L1, 4-1BB, or both, when an antigen-antibody reaction is detected in a biological sample or the level of an antigen-antibody reaction in a biological sample is higher than in a normal sample.

Disease associated with PD-L1, 4-1BB, or both may be due to activation (eg, abnormal activation or overactivation) and/or overproduction (overexpression) of PD-L1, 4-1BB, or both and another. For example, the disease may be cancer, as described above.

In the diagnostic composition and method, the biological sample may be at least one selected from the group consisting of a cell, tissue, biological fluid (e.g., blood, serum, lymph, etc.), etc., obtained (taken) in the patient to make a diagnosis. The normal sample may be at least one selected from the group consisting of a cell, tissue, biological fluid (eg, blood, serum, lymph, urine, etc.), etc., obtained from a patient, not having PD-L1, 4-1BB, or both disease. The patient may be selected from a mammal such as a human. After optional pre-treatment of the sample, the sample can be incubated with the bispecific antibody of the present invention under conditions that allow the antibody to react with the PD-L1 and/or 4-1BB protein potentially present in the sample.

The presence and/or level (concentration) of PD-L1 and/or 4-1BB protein in a sample can be used to identify a patient who is suitable for treatment with a bispecific antibody or a patient who is responsive or receptive to treatment with a bispecific antibody.

In an embodiment, the present invention provides a pharmaceutical composition identifying a patient who is suitable for treatment with a bispecific antibody, or a patient who is responsive or receptive to treatment with a bispecific antibody, wherein the composition contains the bispecific antibody. In another embodiment, the present invention relates to the use of a bispecific antibody to identify a patient who is suitable for treatment with a bispecific antibody, or a patient who is responsive or receptive to treatment with a bispecific antibody. In another embodiment, the present invention provides a method for identifying a patient who is suitable for treatment with a bispecific antibody, or a patient who is responsive or susceptible to treatment with a bispecific antibody, the method comprising contacting a biological sample taken from the patient with the bispecific antibody and detecting antigen-antibody reactions or measurement of the level of antigen-antibody reaction in a biological sample.

In an embodiment, the present invention provides a composition for detecting PD-L1, 4-1BB, or both simultaneously in a biological sample, the composition comprising a bispecific antibody. In another embodiment, the present invention provides a method for detecting PD-L1, 4-1BB, or both simultaneously in a biological sample, the method comprising contacting the biological sample and a bispecific antibody and detecting (measuring) an antigen-antibody reaction (binding) between the bispecific antibody and PD-L1, 4-1BB or both.

In a detection composition and detection method, the term detection of PD-L1, 4-1BB, or both may refer, without limitation, to detecting the presence (and/or absence) and/or level of PD-L1, 4-1BB, or both in a biological sample.

In the detection method, when an antigen-antibody reaction is detected, it can be determined that PD-L1, 4-1BB or both are present in the biological sample, and when an antigen-antibody reaction is not detected, it can be determined that PD-L1, 4 -1BB or both are absent (not present) in the biological sample. Therefore, the detection method may further comprise, after the detection step, determining whether PD-L1, 4-1BB, or both is present in the biological sample when an antigen-antibody reaction is detected, and/or whether PD-L1 is absent (not present), 4-1BB or both in a biological specimen when no antigen-antibody reaction is detected.

In the detection method, the level of PD-L1, 4-1BB, or both can be determined according to the extent of the antigen-antibody reaction (e.g., the amount of antigen-antibody complex formed by the antigen-antibody reaction, the intensity of any signal produced by the antigen-antibody reaction, and etc., which can be measured by any generally accepted means).

The biological sample may comprise at least one sample selected from the group consisting of a cell (eg, tumor cell), tissue (eg, tumor tissue), body fluid (eg, blood, serum, etc.), and the like ., taken from or isolated from a mammal such as a human. The steps of the detection method can be carried out in virto.

In the diagnostic method and/or detection method, the step of detecting an antigen-antibody reaction or measuring the level of an antigen-antibody reaction can be carried out by any conventional method known in the relevant art, such as general enzymatic reactions, fluorescent reactions, luminescent reactions and/or radiation detection. For example, a stage can be

- 31 045275 carried out by a method selected, without limitation, from the group consisting of immunochromatography, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA), fluorescent immunoassay. (FIA), Luminescent Immunoassay (LIA), Western Blot, Microarray, Flow Cytometry, Surface Plasmon Resonance (SPR), etc.

Polynucleotides encoding antibodies and methods for producing antibodies.

In an embodiment, the present invention provides a polynucleotide encoding a bispecific antibody. In particular, according to an embodiment, the present invention relates to a polynucleotide encoding the heavy chain of a bispecific antibody in the form of an IgG-scFv. In another embodiment, the present invention provides a polynucleotide encoding a light chain of a bispecific antibody in the form of an IgG-scFv. The IgG-scFv form may refer to any kind of bispecific antibody comprising a full-length IgG antibody targeting (binding to) one of the PD-L1 and 4-1BB proteins, and a scFv fragment targeting (binding to) the other, where the scFv is bound with the C-terminus and/or N-terminus of a full-length IgG antibody directly (without a peptide linker) or through a peptide linker.

In an embodiment, when the bispecific antibody in the form of an IgG-scFv comprises a full-length anti-PD-L1 IgG antibody, and an anti-4-1BB scFv fragment, the polynucleotide encoding the heavy chain of the bispecific antibody may encode the heavy chain of a full-length anti-PD-L1 IgG antibody , and an anti-4-1BB scFv fragment that is linked to the C-terminus and/or N-terminus of a full-length IgG antibody, directly or through a peptide linker, and a polynucleotide encoding a light chain of a bispecific antibody may encode a light chain of a full-length IgG antibody against PD-L1.

In another embodiment, when the bispecific antibody in the form of an IgG-scFv comprises a full-length anti-4-1BB IgG antibody and an anti-PD-L1 scFv fragment, the polynucleotide encoding the heavy chain of the bispecific antibody may encode the heavy chain of a full-length anti-4-1BB IgG antibody , and an anti-PD-L1 scFv fragment that is linked to the C-terminus and/or N-terminus of a full-length IgG antibody directly or through a peptide linker, and a polynucleotide encoding a light chain of a bispecific antibody may encode a light chain of a full-length IgG antibody against 4 -1BB.

In another embodiment, the present invention provides a recombinant vector comprising a polynucleotide encoding a heavy chain of a bispecific antibody, a polynucleotide encoding a light chain of a bispecific antibody, or both. According to another embodiment, the present invention relates to a recombinant cell transfected with a recombinant vector.

According to another embodiment, the present invention provides a method for producing a bispecific antibody, comprising expressing a polynucleotide encoding a heavy chain of the bispecific antibody, a polynucleotide encoding a light chain of the bispecific antibody, in a cell. The step of expressing the polynucleotide can be carried out by culturing a cell containing the polynucleotide (eg, in a recombinant vector) under conditions allowing expression of the polynucleotide. The method may further comprise isolating and/or purifying the bispecific antibody from the cell culture after the expression or culture step.

Examples

In the following, the present invention is described in more detail by way of examples. The following examples are intended only to illustrate the present invention and are not intended to limit it.

Example 1. Preparation of anti-PD-L1 monoclonal antibodies.

1.1. Production of mouse monoclonal antibodies against human PD-L1 and their analysis.

Mouse monoclonal antibodies against human PD-L1 were constructed using a hybridoma technique as disclosed in international application publication WO 2017-215590.

The amino acid and polynucleotide sequences of the variable regions of the supernatants of the hybridoma, named Hybridoma HL1210-3, are given in table. 5 below.

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Variable sequences of HL1210-3 Table 5 Name Subsequence SEQ ID NO: HL1210-3 VH GAAGTGAAACTGGTGGAGTCTGGGGGAGACTTAG TGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGC AGCCTCTGGATTCACTTTCAGTAGCTATGACATGT CTTGGGTTCGCCAGACTCCGGAGAAGAGTCTGGA GTGGGTCGCAACCATTAGTGATGGTGGTGGTTAC ATCTACTATTCAGACAGTGTGAAGGGGCGATTTA CCATCTCCAGAGACAATGCCAAGAAC AACCTGTA CCTGCAAATGAGCAGTCTGAGGTCTGAGGACACG GCCTTGTATATTTGTGCAAGAGAATTTGGTAAGC GCTATGCTTTGGACTACTGGGGTCAAGGAACCTC AGTCACCGTCTCCTCA 45 HL1210-3 VH EVKLVESGGDLVKPGGSLKLSCAASGFFSSYDMS WVRQTPEKSLEWVATISDGGGYIYYSDSVKGRFTIS RDNAKNNLYLQMSSLRSEDTALYICAREFGKRYAL 46 HL1210-3 VL DYWGQGTSVT GACATTGTGATGACCCAGTCTCACAAATTCATGT CCACATCGGTAGGAGACAGGGTCAGCATCTCCTG CAAGGCCAGTCAGGATGTGACTCCTGCTGTCGCC TGGTATCAACAGAAGCCAGGACAATCTTCCTAAAC TACTGATTTACTCCACATCCTCCCGGTACACTGGA GTCCCTGATCGCTTCACTGGCAGTGGATCTGGGA CGGATTTCACTTTCACCATCAGCAGTGTG CAGGCT GAAGACCTGGCAGTTTATTACTGTCAGCAACATT ATACTACTCCGCTCACGTTCGGTGCTGGGACCAA 47 HL1210-3 VL GCTGGAGCTGAAA DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAW YQQKPGQSPKLLIYSTSSRYTGVPDRFTGSGSGTDF TFTISSVQAEDLAVYYCQQHYTTPLTFGAGTKLELK 48

1.2. Humanization of mouse mAb HL1210-3.

The variable region genes of the monoclonal antibody (mAb) HL1210-3 were used to create a humanized monoclonal antibody through methods widely used in the art and disclosed in international application publication WO 2017-215590.

The amino acid sequences and nucleotide sequences of some of the obtained humanized antibodies are given in table. 6 below.

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Table 6

Humanized antibody sequences (CDR shown in bold)

Name Amino acid sequence SEQ ID NO: HL1210-VH EVKLVESGGDLVKPGGSLKLSCAASGFFSSYDMSW VRQTPEKSLEWVATISDGGGYIYYSDSVKGRFTISRD NAKNNLYLQMSSLRSEDTALYICAREFGKRYALDYW GQGTSVTVSS Hul210 VH.l EVQLVESGGGLVKPGGSLRLSCAASGFFSSYDMSW VRQAPGKGLEWVSTISDGGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREFGKRYALDY ' WGQGTTVTVSS

Hul210 VH.la EVQLVESGGGGLVKPGGSLRLSCAASGFFSSYDMSW

VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCAREFGKRYALD ' YWGQGTTVTVSS

Hul210 VH.lb EVQLVESGGGGLVKPGGSLRLSCAASGFFSSYDMSW

Hul210 VH.2 VRQAPGKSLEWVATISDGGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYICAREFGKRYALDY ' WGQGTTVTVSS EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWI RQAPGKGLEWVSTISDGGGYIYYSDSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCAREFGKRYALDYW GQGTTVTVSS

Hul210 VH.2a EVQLVESGGGGLVKPGGSLRLSCAASGFFSSYDMSWI

RQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCAREFGKRYALDY ' WGQGTTVTVSS

Hu 1210 VH.2b EVQLVESGGGGLVKPGGSLRLSCAASGFFSSYDMSW

VRQAPGKSLEWVATISDGGGYIYYSDSVKGRFTISRD 55 NAKNSLYLQMNSLRAEDTAVYICAREFGKRYALDY

- 34 045275

WGQGTTVTVSS

Hu 1210 VH.3 EVQLLESGGGLVQPGGSLRLSCA ASGFTFSSYDMSW V

RQAPGKGLEWVSTISDGGGYIYYSDSVKGRFTISRDN

SKNTLYLQMNSLRAEDTAVYYCAREFGKRYALDYW '

GQGTTVTVSS

Hul210 VH.3a EVQLLESGGGLVQPGGSLRLSCAASGFFSSYDMSWV RQAPGKSLEWVATISDGGGYIYYSDSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYICAREFGKRYALDYW 57 Hul210 VH.4 GQGTTVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKGLEWVSTISDGGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYYCAREFGKRYALDY 58 Hul210 VH.4a WGQGTTVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR 50 DNAKNSLYLQMNSLRDEDTAVYYCAREFGKRYALD YWGQGTTVTVSS Hul210 VH.4b EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISDGGGYIYYSDSVKGRFTISRD A.O. NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY WGQGTTVTVSS Hul210 VH.4c EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISEGGGYIYYSDSVKGRFTISRD A1 NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY WGQGTTVTVSS Hu 1210 VH.4d EVQLVESGGGGLVQPGGSLRLSCAASGFFSSYDMSW (H12 VH) VRQAPGKSLEWVATISDAGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY Hul210 VH.4c WGQGTTVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISDVGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY WGQGTTVTVSS 63

- 35 045275

Hu 1210 VH.5 EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCAREFGKRYALD YWGQGTLVTVSS

HU 1210 VH.5a EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYICAREFGKRYALD ' YWGQGTLVTVSS

HU 1210 VH.5b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSW VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR

DNAKNSLYLQMNSLRAEDTAVYICAREFGKRYALD

YWGQGTTVTVSS

HU 1210 EVQLVESGGGGLVQPGGSLRLSCAASGFFSSYDMSW

VH.5C VRQAPGKGLEWVATISDGGGYIYYSDSVKGRFTISR

DNAKNNLYLQMNSLRAEDTAVYICAREFGKRYALD YWGQGTLVTVSS

HU 1210 VH.5d EVQLVESGGGGLVQPGGSLRLSCAASGFFSSYDMSW

VRQTPEKSLEWVATISDGGGYIYYSDSVKGRFTISRD 68

NAKNNLYLQMNSLRAEDTAVYICAREFGKRYALDY WGQGTLVTVSS

HL1210-VK DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWY

QQKPGQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTFTI 69 SSVQAEDLAVYYCQQHYTTPLTFGAGTKLELK

Hul210 VK.l DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ (H12VL) QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS 70

S LQPEDIATYYCQQHYTTPLTFGQGTKLEIK

Hu 1210 VL. 1 a DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ QKPGKSPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS 71 S LQPEDIATYYCQQHYTTPLTFGQGTKLEIK

Hul210 VK.2 DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTLTIS 72 SLQPEDFATYYCQQHYTTPLTFGQGTKLEIKR

Hul210 VK.2a DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ 73

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QKPGKAPKLLIYSTSSRYTGVPDRFTGSGSGTDFTLTI SSLQPEDFATYYCQQHYTTPLTFGQGTKLEIKR Hul210 VK.2b DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ QKPGQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTLTIS SLQPEDFATYYCQQHYTTPLTFGQGTKLEIKR 74 Hul210 VK.2c DIQMTQSPSSLSSASVGDRVTISCKASQDVTPAVAWYQ QKPGQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTLTIS SLQPEDFATYYCQQHYTTPLTFGQGTKLEIKR 75 Name Nucleic acid sequence SEQ ID NO: HL1210 VH GAGGTGAAGCTGGTGGAGAGCGGCGGAGATCGGT GAAGCCTGGCGGCAGCCTGAAGCTGAGCTGTGCCG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGGCAGACCCCCGAGAAGAGCCTGGAGTGG GTGGCCACCATCAGCGATGGCGGCGGCTACATCTA CTACAGCGACAGCGTGAAGGGCAGGTTCACCATCA GCAGGGACAACGCCAAGAACAACCTGTACCTGCAG ATGAGCAGCCTGAGGAGCGAGGACACCGCCCTGTA CATCTGCGCCAGGGAGTTCGGCAAGAGGTACGCCC TGGACTACTGGGGACAGGGCACCAGCGTGACCGTG AGCAGC 76 Hul210 VH.l GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGAGCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACGCCAAGAACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC 77 Hul210 VH.la GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG 78

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CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCA AAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA

GCAGC

Hu 1210 VH. 1 b GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT

GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 79 CAGGGACAACGCCAAGAACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAG GACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.2 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGAGCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 8 0 CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.2a GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT

GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG 81

CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT

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GGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG Hul210 VH.2b CAGGGACAACGCAAGAACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GAAGCCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 82 Hul210 VH.3 CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC GAGGTGCAGCTGCTGGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGAGCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 83 Hul210 VH.3a CAGGGACAACAGCAAGAACACCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC GAGGTGCAGCTGCTGGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG 84

- 39 045275

GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACAGCAAGAACACCCTGTACCTGCAGA TGAACAGCCTGAGGGCCGAGCACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.4 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGAGCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 8 5 CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGATGAGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.4a GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT

GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 86 CAGGGACAACGCCAAGAACAGCCTGTACCTGCAGA TGAACAGCTGAGGGATG AGGACACCGCCGTGTAC TACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.4b GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT

GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT 87 GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGATGGCGGCGGCTACATCTAT

- 40 045275

Hul210 VH.4c TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACGCCAAGAACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGATGAGGACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGAAGGCGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGATGAGGACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAA GGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA 88 GCAGC Hul210 VH.4d GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGATGCGGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG CAGGGACAACGCCAAGAACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGATGAGGACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC 89 Hul210 VH.4c GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGT GCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTG CCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCT GGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGG GTGGCCACCATCTCCGATGTTGGCGGCTACATCTAT TACTCCGACAGCGTGAAGGGCAGGTTCACCATCAG 90

- 41 045275

CAGGGACAACGCCAAGACAGCCTGTACCTGCAGA TGAACAGCCTGAGGGATGAGGACACCGCCGTGTAC ATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCT GGACTACTGGGGCCAGGGCACAACCGTGACCGTGA GCAGC

Hu 1210 VH.5 GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGT GCAACCTGGAGGCTCCCTGAGGCTGTCCTGTGCCGC TTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTG GGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGG TGGCCACCATCTCCGACGGAGGCGGCTACATCTACT ACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCC 91 GGGACAACGCCAAGAACTCCCTGTACCTGCAGATG AACTCTCTCAGGGCTGAGGACACCGCCGTGTATTAC TGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGA TTACTGGGGCCAGGGCACACTGGTGACAGTGAGCT cc

Hul210 VH.5a GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGT GCAACCTGGAGGCTCCCTGAGGCTGTCCTGTGCCGC TTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTG GGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGG TGGCCACCATCTCCGACGGAGGCGGCTACATCTACT ACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCC 92 GGGACAACGCCAAGAACTCCCTGTACCTGCAGATG AACTCTCTCAGGGCTGAGGACACCGCCGTGTATATC TGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGA TTACTGGGGCCAGGGCACACTGGTGACAGTGAGCT CC

Hu 1210 VH.5b GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGT GCAACCTGGAGGCTCCCTGAGGCTGTCCTGTGCCGC TTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTG GGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGG 93 TGGCCACCATCTCCGACGGAGGCGGCTACATCTACT ACTCCGACTCCGTGAAGGGCAGGTTCAC CATCTCCC GGGACAACGCCAAGAACAACCTGTACCTGCAGATG

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AACTCTCTCAGGGCTGAGGACACCGCCGTGTATATC

TGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGA TTACTGGGGCCAGGGCACACTGGTGACAGTGAGCT cc

Hu 1210 VH.5c GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGT

GCAACCTGGAGGCTCCCTGAGGCTGTCCTGTGCCGC TTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTG GGTGAGGCAGACCCCTGAGAAGAGCCTGGAGTGGG TGGCCACCATCTCCGACGGAGGCGGCTACATCTACT ACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCC 94 GGGACAACGCCAAGAACAACCTGTACCTGCAGATG AACTCTCTCAGGGCTG AGGACACCGCCGTGTATATC TGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGA TTACTGGGGCCAGGGCACACTGGTGACAGTGAGCT CC

Hul210 VH.5d GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGT GCAACCTGGAGGCTCCCTGAGGCTGTCCTGTGCCGC TTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTG GGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGG TGGCCACCATCTCCGACGGAGGCGGCTACATCTACT ACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCC 95 GGGACAACGCCAAGAACTCCCTGTACCTGCAGATG AACTCTCTCAGGGCTGAGGACACCGCCGTGTATATC TGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGA TTACTGGGGCCAGGGCACAACCGTGACAGTGAGCT CC

HL 1210 VK GACATCGTGATGACCCAGAGCCACAAGTTCATGAG

CACCAGCGTGGGCGATAGGGTGAGCATCAGCTGCA

AGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGG

TACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCT

GATCTACAGCACCAGCAGCAGGTACACCGGCGTGC

CCGACAGGTTCACAGGAAGCGGCAGCGGCACCGAC TTCACCTTCACCATCAGCAGCGTGCAGGCCGAGGA CCTGGCCGTGTACTACTGCCAGCAGCACTACACCAC

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CCCTCTGACCTTCGGCGCCGGCACCAAGCTGGAGCT GAAG Hul210 VK.l GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAG CGCTAGCGTGGGCGACAGGGTGACCATCACCTGCA AGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGG TACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCT GATCTACAGCACCAGCAGCAGGTACACCGGCGTGC 97 CCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGAC TTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGAC ATCGCCACCTACTACTGCCAGCAGCACTACACCACC CCTCTGACCTTCGGCCAGGGCACCAAGCTGGAGAT CAAG Hul210 VK.la GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAG CGCTAGCGTGGGCGACAGGGTGACCATCACCTGCA AGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGG TACCAGCAGAAGCCCGGCAAGTCCCCCAAGCTGCT GATCTACAGCACCAGCAGCAGGTACACCGGCGTGC 98 CCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGAC TTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGAC ATCGCCACCTACTACTGCCAGCAGCACTACACCACC CCTCTGACCTTCGGCCAGGGCACCAAGCTGGAGAT CAAG Hul210 VK.2 GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCC GCTTCCGTGGGCGACAGGGTGACCATCACCTGCAA GGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGT ATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTG ATCTACAGCACATCCTCCCGGTACACCGGAGTGCCC 99 TCCAGGTTTAGCGGCAGCGGCTCCGGCACCGATTTC ACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTC GCCACCTACTACTGCCAGCAGCACTACACCACACCCC CTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAA GCGG Hul210 VK.2a GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCC 100 GCTTCCGTGGGCGACAGGGTGACCATCACCTGCAA

- 44 045275

GGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGT ATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTG ATCTACAGCACATCCTCCCGGTACACCGGAGTGCCC GACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTC ACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTC GCCACCTACTACTGCCAGCAGCACTACACCACACCC CTGACCTTCGGCCAGGGCACCAAGC TGGAGATCAA

GCGG

Hu 1210 VK.2b GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCC GCTTCCGTGGGCGACAGGGTGACCATCACCTGCAA

GGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGT ATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTG ATCTACAGCACATCCTCCCGGTACACCGGAGTGCCC 101

GACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTC ACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTC GCCACCTACTACTGCCAGCAGCACTACACCACACCC CTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAA GCGG

Hul210 VK.2c GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCC GCTTCCGTGGGCGACAGGGTGACCATCAGCTGCAA GGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGT ATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTG ATCTACAGCACATCCTCCCGGTACACCGGAGTGCCC 102

GACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTC ACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTC

GCCACCTACTACTGCCAGCAGCACTACACCACACCCC

CTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAA GCGG

Humanized VH and VK genes were produced synthetically and then respectively cloned into vectors containing human gamma-1 and human kappa constant domains. Pairing of human VH and human VK resulted in the creation of 40 humanized antibodies (see Table 7).

Table 7

Humanized antibodies with their VH and VL regions

VH VK Ni1210 VH.1 Ni1210 VH.la Ni1210 VH.lb Ni1210 VH.2 Ni1210 VH.2a Ni1210 VH 2.b Hul210 VH Neither 1210 VK.1 Ni1210-1 Ni1210-2 Ni1210-3 Ni1210-4 Ni1210-5 Ni1210 VK.la Ni1210-7 Ni1210-8 Ni1210-9 Ni1210-10 Ni1210-11 Neither 1210 VK H1210 Chimera

VH VK Hul210 VH.3 Hul210 VH.3a Hul210 VH.4 Hul210 VH.4a Hul210 VH.4b Hu 1210 VK.l Hu1210-13 Hul210-14 Hul210-15 Hul210-16 Hul210-17 Hul210 VK.la Hul210-18 Hul210-19 Hul210-20 Hul210-21 Hul210-22

- 45 045275

VH VK Hul210 VH.5 HU1210 VH.5a HU1210 VH.5b HU1210 VH.5c HU1210 VH.5d Ni1210 VK.2 Hul210-23 Hul210-27 Hul210-31 Hul210-32 Hul210-36 Ni1210 VK.2a Hul210-24 Hul210-28 Hul210-33 Hul210-37 Hul210 VK.2b Hu 1210-25 Hu 1210-29 Hul210-34 Hul210-38 Hul210 VK.2c Hul210-26 Hul210-30 Hul210-35 Hul210-39

VH VK Hul210 VH.4c Hul210 VH.4d Hul210 VH.4e Hul210 VK.l Hul210-40 Hul210-41 Hul210-42

1.3. PD-L1 epitope identification.

This study was conducted to identify amino acid residues involved in the binding of PD-L1 to the antibody of the present invention.

An alanine scan library for PD-L1 was constructed. Briefly, 217 PD-L1 mutant clones were generated on an integrated molecular protein engineering platform. The binding of Fab Hu1210-41 to each variant in the PD-L1 mutation library was determined in duplicate using high-throughput flow cytometry. Background fluorescence was subtracted from each raw data point and normalized to reactivity with wild-type (WT) PD-L1. For each PD-L1 variant, the mean binding value was plotted as a function of expression. To define preliminary critical clones (circles with crosses), cutoff values (dashed lines) of >70% of wild-type binding to a control monoclonal antibody (MAb) (Mab MIH1, generated in the laboratory) and <30% of wild-type reactivity to Fab were used Hul210-41 (Fig. 3). Y134, K162, and N183 residues of PD-L1 were identified as essential residues for Hu1210-41 binding. The low reactivity of clone N183A with Fab Hu1210-41 suggests that it is the major energetic contributor to Hu1210-41 binding, with minor contributions from Y134 and K162.

Critical residues (spheres) were identified on the 3D structure of PD-L1 as shown in FIG. 4. These residues, Y134, K162 and N183, thus constitute the PD-L1 epitopes responsible for binding to antibodies according to various embodiments of the present disclosure.

It is interesting to note that Y134, K162, and N183 are all located within the IgC domain of the PD-L1 protein. The extracellular portions of both PD-1 and PD-L1 have an IgV domain and an IgC domain. It is well known that PD-L1 binds to PD-1 through binding between their IgV domains. However, unlike such conventional antibodies, Hu1210-41 binds to an IgC domain that would be expected to be ineffective in inhibiting PD-1/PD-L1 binding. This different Hu1210-41 epitope, surprisingly, likely contributes to the distinct activities of Hu1210-41.

1.4. Antibody engineering for anti-PD-L1 antibody.

Examples 1.4 attempted to identify further improved Hu1210-41-based antibodies using mutagenesis.

Four sub-libraries were constructed for antibody engineering of anti-PD-L1 monoclonal antibody using any of the following strategies. In strategy 1, mutagenesis of the CDR3 heavy chain variable domain of VH or VL-CDR3 was accomplished by highly random mutation. In strategy 2, two combination CDR libraries consisting of (VH-CDR3, VL-CDR3, and VL-CDR1) or (VH-CDR1, VH-CDR2, and VL-CDR2) were generated by CDR walks with controlled mutation rates.

Bio-panning. Phage panning methods have been adapted by reducing the incubation/binding time before stringent washing conditions. Briefly, 100 μl of magnetic streptavidin beads (Invitrogen, USA) were blocked with 1 ml of MPBS for 1 h at room temperature. In another tube, the phage library was preincubated (5x10 ^L 11~12 per cycle) with 100 μl magnetic streptavidin beads in 1 ml MPBS to remove unwanted binders. A magnetic particle concentrator was used to separate the phage and beads. Biotinylated PD-L1 protein was added to the phage and incubated for 2 h at room temperature and mixed gently using an overhead shaker. Beads carrying phage from solution were separated in a magnetic particle concentrator, and the supernatant was discarded. The beads were washed with fresh wash buffer, ten times with PBST and ten times with PBS (pH 7.4). 0.8 ml of 0.25% trypsin in PBS (Sigma, USA) was added and incubated for 20 min at 37°C to elute the phage. The resulting phage was titrated and excluded from the next panning cycle, decreasing the antigen concentration from cycle to cycle.

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ELISA screening and ranking on/off

Clones were selected and induced based on the desired panning result, phage ELISA was performed for primary screening, positive clones were analyzed by sequencing, and unique hotspots were found.

In table Figure 8 shows the identified mutations. As shown below, hotspot mutation residues and/or their surrogates are underlined.

Table 8

Mutations in CDR

CDR-H1 (SEQ ID No.) CDR-H2 (SEQ ID No.) CDR-H3 (SEQ ID No.) WT (H12) SYDMS (107) TISDAGGYIYYSDSVKG (117) EFGKRYALDY (127) vz SYDMS (108) TISDAGGYIYYRDSVKG (118) EFGKRYALDY (128) C4 SYDMS (109) TISDAGGYIYYRDSVKG (119) EFGKRYALDS (129) IN 1 SYDMS (110) TISDAGGYIYYRDSVKG (120) EIFNRYALDY (130) Wb SYDMS (111) TISDAGGYIYYRDSVKG (121) ELPWRYALDY (131) nw SYDMS (112) TISDAGGYIYYRDSVKG (122) ELHFRYALDY (132) C6 SYDMS (113) TISDAGGYIYYRDSVKG (123) ELYFRYALDY (133) A1 SYDMS (114) TISDAGGYIYYRDSVKG (124) ELLHRYALDY (134) A2 SYDMS (115) TISDAGGYIYYRDSVKG (125) ELRGRYALDY (135) AZ SYDMS (116) TISDAGGYIYYRDSVKG (126) EFGKRYALDY (136) CDR-L1 (SEQ ID No.) CDR-L2 (SEQ ID No.) CDR-L3 (SEQ ID No.) W.T. KASQDVTPAVA (137) STSSRYT (147) QQHYTTPLT (157) vz KAKQDVTPAVA (138) STSSRYT (148) MQHYTTPLT (158) C4 KASQDVWPAVA (139) STSSRYT (149) QQHSTTPLT (159) IN 1 KASQDVTPAVA (140) STSSRYT (150) QQHYTTPLT(160) Wb KASQDVTPAVA (141) STSSRYT (151) QQHYTTPLT (161) nw KASQDVTPAVA (142) STSSRYT (152) QQHYTTPLT (162) C6 KASQDVTPAVA (143) STSSRYT (153) QQHYTTPLT (163) A1 KASQDVTPAVA (144) STSSRYT (154) QQHYTTPLT (164) A2 KASQDVTPAVA (145) STSSRYT (155) QQHYTTPLT (165) AZ KASQDVTPAVA (146) STSSRYT (156) QQHSDAPLT (166)

The amino acid sequences of the variable regions of these antibodies are shown in table. 9 below.

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Table 9 Antibody sequences

Name Sequence SEQ ID NO:

WT-VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSWV 167

RQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRDNA KNSLYLQMNSLRDEDTAVYICAREFGKRYALDYWGQ GTTVTVSS

WT-VK DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQQ 168

KPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTISSL

QPEDIATYYCQQHYTTPLTFGQGTKLEIK

B3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSS YDMSWV 169

RQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRDNA KNSLYLQMNSLRDEDTAVYICAREFGKRYALDYWGQ GTTVTVSS

B3-VK DIQMTQSPSSLSSASVGDRVTITKAKQDVTPAVAWYQ 170

QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTTSS LQPEDIATYYCMQHYTTPLTFGQGTKLEIK

C4-VH EVQLVESGGGGLVQPGGSLRLSCAASGFFSSYDMSWV 171

RQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRDNA KNSLYLQMNSLRDEDTAVYICAREFGKRYALDSWGQG TTVTVSS

C4-VK DIQMTQSPSSLSASVGDRVTITCKASQDVWPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFT1SS 172 LQPEDIATYYCQQHSTTPLTFGQGTKLEIK

B1 -VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW

VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD

NAKNSLYLQMNSLRDEDTAVYICAREIFNRYALDYW

GQGTTVTVSS

Bl-VK DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFT1S 174 SLQPEDIATYYCQQHYTTPLTFGQGTKLEIK

B6-VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW

VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICARELPWRYALDY WGQGTTVTVSS

B6-VK DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS 176 SLQPEDIATYYCQQHYTTPLTFGQGTKLEIK

C3-VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW

VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICARELHFRYALDYW GQGTTVTVSS

C3-VK DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ 178

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C6-VH QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS SLQPEDIATYYCQQHYTTPLTFGQGTKLEIK EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICARELYFRYALDYW GQGTTVTVSS 179 C6-VK DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS SLQPEDIATYYCQQHYTTPLTFGQGTKLEIK 180 A1-VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD 1 21 A1-VK NAKNSLYLQMNSLRDEDTAVYTCARELLHRYALDYW GQGTTVTVSS DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS 182 A2-VH SLQPEDIATYYCQQHYTTPLTFGQGTKLEIK EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVAT1SDAGGY1YYRDSVKGRFT1SRD NAKNSLYLQMNSLRDEDTAVYICARELRGRYALDY WGQGTTVTVSS 183 A2-VK DIQMTQSPSSLSSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS S LQPEDIAT YYCQQH YTTPLTFGQGTK LEIK 184

AZ-VH EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW

VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFT1SRD NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY '

WGQGTTVTVSS

A3-VK DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTIS 186 SLQPEDIATYYCQQHSDAPLTFGQGTKLEIK

Table 10

Heavy chain variable regions for clones H12 and B6

Antibody no. VH SEQ ID NO: H12 EVQLVESGGGLVQPGGSLRLSCAASGFFSSYDMSW VRQAPGKSLEWVATISDAGGYIYYSDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICAREFGKRYALDY WGQGTTVTVSS 103 Wb EVQLVESGGGLVQPGGSLRLSCAASGFFSYDMSW VRQAPGKSLEWVATISDAGGYIYYRDSVKGRFTISRD NAKNSLYLQMNSLRDEDTAVYICARELPWRYALDY WGQGTTVTVSS 104

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Table 11 Light chain variable regions for clones H12 and B6 Antibody no. VL SEQ ID NO: H12 DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTISS LQPEDIATYYCQQHYTTPLTFGQGTKLEIK 105 Wb DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQ QKPGKAPKLLIYSTSSRYTGVPSRFSGSGSGTDFTFTISS LQPEDIATYYCQQHYTTPLTFGQGTKLEIK 106

1.5. Protein kinetics for PD-L1.

In order to examine the binding kinetics of the humanized antibody, affinity ranking was performed in this example using Biacore. As shown in table. 12 below, H12 and B6.

Table 12 Antibody KD(M) kon(l/Mc) kdis(l/c) Chi H12 6.122E-09 7.124E+04 4.361E-04 0.0415 Wb 4.248E-09 9.827E+04 4.175E-04 0.0766

As shown in table. 12, the anti-PD-L1 antibodies tested show high binding affinities to PD-L1.

Example 2. Preparation of anti-4-1BB monoclonal antibodies.

2.1. Screening of complete human monoclonal antibodies against 4-1BB Panning of a phage library in an immunotube for 4-1BB.

To pan the library against target molecules, a total of four rounds of panning were performed using 4-1BB coated immunotubes.

Bacterial colonies as a result of 3 cycles of panning were grown in SB-carbenicillin in a 96-well plate until cloudy, after which 1011 PFU of the helper phage VCSM13 were added to each well. After 1 h of infection at 37°C with gentle shaking (80 rpm), 70 μg/ml kanamycin was added, and cells were cultured overnight at 30°C with shaking at 200 rpm.

The next day, the plates were centrifuged and supernatants containing phages were added to 4-1BB antigen-coated ELISA plates blocked with 3% BSA in PBST. After 1 h of incubation at room temperature, the plates were washed three times with PBST and anti-M13 antibody was added. The plates were incubated for 1 h, washed three times with PBST, and binding activity was measured using tetramethylbenzidine (TMB).

Specific 4-1BB binders were amplified for plasmid DNA sequencing. Ig light chain V genes (VL) and VH sequences were analyzed to identify unique sequences and determine sequence diversity.

Table 13

Heavy chain variable regions

Antibody no. VH S.E. QID NO: 41B01 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYDMSWVRQAPGK GLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDGQRNSMREFDYWGQGTLVTVSS 187 41B01.01 EVQLLESGGGLVQPGGSLRLSCAASGFFSSYDMSWVRQAPGK GLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS 188

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41В01.02 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGK GLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS 189 41VO1.OZ EVQLLESGGGLVQPGGSLRLSCAASGFFSSYDMSWVRQAPGK GLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS 190 41В01.04 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGK GLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARDAQRQSMREFDYWGQGTLVTVSS 191 41В02 EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYDMSWVRQAPGK GLEWVSVIYPDDGNTYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDAAVYYCAKHGGQKPTTKSSSAYGMDGWGQGTLVTVSS 192

Table 14

Heavy chain CDR

Antibody No. CDR H1 S.E. QID NO: CDR H2 S.E. QID NO: CDR H3 S.E. QID NO: 41В01 SYDM S 10 WISYSGGSIYYADS VKG 12 DGQRNSMREFDY 14 41В01.01 SYDM S 10 WISYSGGSIYYADS VKG 12 DAQRNSMREFDY 16 41В01.02 SYDM S 10 WISYSGGSIYYADS VKG 12 DAQRQSMREFDY 17 41В01.03 SYDM s 10 WISYSGGSIYYADS VKG 12 DAQRNSMREFDY 16 41В01.04 SYDM s 10 WISYSGGSIYYADS VKG 12 DAQRQSMREFDY 17 41В02 G.Y.D.M. S eleven VIYPDDGNTYYAD SVKG 13 HGGQKPTTTKSSS AYGMDG 15

Table 15

Light chain variable regions

Antibody no. VL SEQ ID NO: 41B01 QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 193 41B01.0 1 QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 194 41B01.0 2 QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 195 41B01.0 3 QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 196

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41В01.0 4 QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 197 41В02 QSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGT APKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCATWDYSLSGYVFGGGTKLTVL 198

Table 16

Light chain CDR

Antibody no. CDRL1 S.E. QID NO CDR L2 S.E. QID NO: CDR L3 S.E. QID NO: 41В01 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20 41В01.01 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20 41В01.02 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20 41В01.03 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20 41В01.04 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20 41В02 SGSSSNIGNNYVT 18 ADSHRP S 19 ATWDYSLS GYV 20

2.2. Antigen-binding abilities of anti-4-1BB antibodies with human 4-1BB.

(1) Antigen binding measured by ELISA.

To evaluate antigen binding activity, candidate antibodies were subjected to ELISA analysis. Briefly, microtiter plates were coated with human 4-lBB-Fc protein at a concentration of 0.1 μg/ml in PBS, 100 μl/well at 4°C overnight, then blocked with 100 μl/well 5% BSA. Five-fold dilutions of humanized antibodies {41B01 and 41B02}, starting at 10 μg/ml, were added to each well and incubated for 1-2 hours at room temperature. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody for 1 h at room temperature. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-630 nm. As shown in FIG. 5, the anti-4-1BB antibodies tested demonstrated the ability to bind to 4-1BB.

(2) Cellular binding measured by FACS.

In order to evaluate antigen binding property, candidate antibodies were analyzed for their binding to expressed mammalian 4-1BB protein by FACS. Briefly, 4-lBB-Jurkat cells were incubated with antibodies (41B01 and 41B02). After washing with FACS buffer (1% BSA in PBS), FITC anti-human IgG antibody was added to each well and incubated at 4°C for 1 hour. The MFI for FITC was assessed by FACS Caliber. As shown in FIG. 6, the anti-4-1BB antibodies tested exhibit the ability to bind to 4-1BB, which is expressed on the cell surface and can effectively bind to 4-1BB expressed on mammalian cells.

(3) Protein kinetics for 4-1BB.

To examine the binding kinetics of the humanized antibody, affinity ranking was performed in this example using Octet Red 96. As shown in Table. 17 below, 41В01 and 41В02.

Table 17

Antibody KD(M) kon(l/Mc) kdis( 1/c) Chi R ² 41B01 1.80E-10 6.58E+05 1.19E-04 0.0392 0.9987 41B02 1.01E-09 5.95E+05 6.03 E-04 0.0525 0.9973

As shown in table. 17, Anti-4-1BB antibodies tested demonstrate high affinity

-52045275 binding ability to 4-1BB.

Example 3. Preparation of aHTu-PD-L1/aHTu-4-1BB bispecific antibodies.

As an example, clones Hu1210-41 (Hu1210 VH.4dxHu1210 VK.1, see Table 6, hereinafter H12) and B6 (see Table 12) were selected among the anti-PD-L1 clones obtained in example 1, clones 41B01, 41B01.01, 41B01.02, 41B01.03, 41B01.04 and 41B02 among the anti-4-1BB clones obtained in example 2, to obtain an anti-PD-L1/anti-4-1BB bispecific antibody in the form of scFv Full length IgG X. When PD-L1 was placed in the whole IgG portion, an ADCC backbone truncated mutant IgG1 (N297A mutation, US Pat. No. 7332581, 8219149, etc.) was used, and when 4-1BB was placed in the whole IgG portion, IgG4 was used.

DNA segment 1, having the nucleotide sequence encoding the heavy chain of the anti-PD-L1/anti-4-1BB bispecific antibody IgG antibody, was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and DNA segment 2, having the nucleotide sequence encoding anti-PD-L1/anti-4-1BB bispecific antibody IgG light chain was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2). DNA segment 3 encoding the scFv was then fused into a portion of DNA segment 1 corresponding to the C-terminus of the Fc region of the IgG antibody inserted into plasmid 1 using DNA segment 4 encoding a 10 amino acid long linker peptide consisting of (GGGGS )2, for the construction of vectors for the expression of bispecific antibodies. In addition, to stabilize the scFv, an additional modification was used to create a disulfide bridge fusing VL103-VH44 to the C-terminus of the light chain and the C-terminus of the heavy chain, respectively.

The sequences of the heavy chain, light chain, scFv and DNA segments are given in table. 18.

Table 18 Bispecific antibody containing anti-PD-L1 clone in IgG form and anti-4-1BB clone in scFv form (PD-L1x4-1BB)

ABLPNB.01 ; (bispecific antibody containing anti-PD-Ll H12 clone in IgG form and anti-41 BB 41 Β0Ί clone in scFv form)

Amino acid Nucleotide sequence sequence (5’—>3’) (N’^C’) (Seq ID (SeqIDNo.) No.) Heavy Heavy chain EVQLVESGGGGLVQ GAGGTGCAGCTGGTGGAGA ; component H12 PGGSLRLSCAASG GCGGAGGAGGACTGGTGCA FTFSSYDMSWVRQ ACCCGGAGGCAGCCTGAGA APGKSLEWVATIS CTGAGCTGCGCTGCCAGCGG DAGGYIYYSDSVK CTTCACCTTCAGCAGCTACG GRFTISRDNAKNS ACATGAGCTGGGTGAGACA LYLQMNSLRDEDT GGCCCCTGGCAAAAGCCTGG AVYICAREFGKRY AGTGGGTGGCCACCATCTCC ALDYWGQGTTVT GATGCGGGCGGCTACATCTA VSSASTKGPSVFPL TTACTCCGACAGCGTGAAGG APSSKSTSGG TAA GCAGGTTCACCATCAGCAGG LGCLVKDYFPEPV GACAACGCCAAGAACAGCC TVSWNSGALTSGV TGTACCTGCAGATGAACAGC HTFPAVLQSSGLY CTGAGGGATGAGGACACCG SLSSVVTVPSSSLG CCGTGTACATCTGCGCCAGG TQTYICNVNHKPS GAGTTCGGCAAAAGGTACG NTKVDKKVEPKSC CCCTGGACTACTGG GGCCAG

- 53 045275

DKTHTCPPCPAPEL GGCACAACCGTGACCGTGA LGGPSVFLFPPKPK GCAGCgctAgcAccAAgGGCCC DTLMISRTPEVTCV CTCTGTGTTCCCTCTGGCCCC VVDVSHEDPEVKF TTCCTCTAAATCCACCTCTG NWYVDGVEVHNA GCGGAACCGCTGCTCTGGGC KTKPREEQYASTY TGTCTGGTCAAGGACTACTT RVVSVLTVLHQD CCCTGAGCCCGTGACCGTGT WLNGKEYKCKVS CTTGGAATTCTGGCGCTCTG NKALPAPIEKTISK ACCAGCGGAGTGCACACCTT AKGQPREPQVYTL TCCAGCTGTGCTGCAGTCCT PPSREEMTKNQVS CCGGCCTGTACTCTCTGTCC LTCLVKGFYPSDIA TCTGTCGTGACAGTGCCTTC VEWESNGQPENN CAGCTCTCTGGGCACCCAGA YKTPPPVLDSDGS CCTACATCTGCAACGTGAAC FFLYSKLTVDKSR CACAAGCCCTCCAACACCAA WQQGNVFSCSVM GGTGGACAAGAAGGTGGAA HEALHNHYTQKSL CCCAAGTCCTGCGACAAGAC SLSPGK CCACACCTGTCCTCCATGTC (199) CTGCTCCAGAACTGCTGGGC Linker GGGGSGGGGSGG GGACCCTCCGTGTTCCTGTT GGS (200) CCCTCCAAAGCCTAAGGACA scFv VL CCCTGATGATCTCCCGGACC 41B01 QSVLTQPPSASGTP CCTGAAGTGACCTGCGTGGT GRRVTISSCSGSSSN GGTGGATGTGTCCCACGAGG IGNNYVTWYQQL ATCCCGAAGTGAAGTTCAAT PGTAPKLLIYADS TGGTACGTGGACGCGTGGA HRPSGVPDRFSGS AGTGCACAACGCCAAGCC KSGTSASLAISGLR AAGCCTAGAGAGGAACAGT SEDEADYYCATW ACgccTCCACCTACCGGGTGG DYSLSGYVFGCGT TGTCCGTGCTGACCGTTCTG KLTVL (201) CACCAGGATTGGCTGAACGG Linker GGGGSGGGGSGG CAAAGAGTACAAGTGCAAG GGSGGGGS (202) GTGTCCAACAAGGCCCTGCC VH EVQLLESGGGLVQ TGCCCCTATCGAAAAGACC A PGGSLRLSCAASG TCTCTAAGGCCAAGGGCCAG FTFSSYDMSWVRQ CCCCGGGAACCTCAAGTGTA APGKCLEWVSWIS CACCTTGCCTCCCAGCCGGG YSGGSIYYADSVK AAGAGATGACCAAGAACCA GRFTISRDNSKNTL GGTGTCCCTGACCTGCCTGG YLQMNSLRAEDTA TTAAGGGCTTCTACCCCTCC VYYCARDGQRNS GATATCGCCGTGGAATGGGA MREFDYWGQGTL GTCTAACGGCCAGCCCGAGA VTVSS ACAACTACAAGACCACCCCT (203) CCTGTGCTGGACTCCGACGG

CTCATTCTTCCTGTACTCCAA

GCTGACCGTGGACAAGTCTC

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GGTGGCAGCAGGGCAACGT

GTTCTCCTGCTCTGTGATGC ACGAGGCCCTGCACAACCAC TACACCCAGAAGTCCCTGTC CCTGTCTCCCGGCAAAGGTG GGGGGGGATCTGGTGGTGGT GGATCAGGGGGTGGGGGGT CTCAAAGCGTACTCACCCAA CCTCCATCTGCATCCGGTAC ACCTGGTCGGCGAGTAACCA TCTCCTGCTCTGGGAGCTCT TCTAATATTGGTAACAACTA TGTCACCT GGTATCAGCAGT TGCCTGGGACAGCACCCAAA CTTCTTATATATGCCGATAG CCATCGGCCTTCCGGCGTAC CCGATCGCTTCTCCGGGTCA AAATCTGGAACATCTGCCTC ACTCGCAATTAGTGGATTGC GATCTGAGGATGAAGCAGA TTATTATTGCGCTACCTGGG ATTATTCACTTTCTGGCTAC GTCTTTGGTtgtggaACAAAAC TTACCGTGTTGGGC GGCGGA GGAAGCGGAGGCGGCGGTT CTGGTGGTGGCGGTAGCGGA GGTGGTGGATCTGAAGTACA GCTTCTTGAGTCTGGCGGAG GATTGGTCCAGCCAGGCGGT TCCCTCCGCCTGTCATGTGC CGCATCCGGCTTTACTTTCTC TAGTTATGATATGAGCTGGG TTCGCCAAGCTCCTGGCAAA tgcCTGGAGTGGGTCTCCTGG atttcatactcagg tggcag

- 55 045275

CATCTATTATGCTGACAGTG

Lightweight Light chain Η12 component

TGAAAGGTCGCTTTACAATC TCCCGAGATAACAGCAAAA ACACCTTGTACCTGCAAATG AACAGCCTTCGCGCAGAGG ACACAGCCGTATATTATTGC GCTCGCGATGGACAACGTAA TTCTATGCGTGAGTTTGACT ACTGGGGACAGGGGACATT GGTCACTGTATCTTCCtga (204)

DIQMTQSPSSLSAS GACATCCAGATGACCCAGA

VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQ GTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACT ACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (28) ATCAAGAGAACCGTGGCCG

CTCCCTCCGTGTTCATCTTCC

CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TTCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC

- 56 045275

TCCCAAGAGTCTGTGACCGA

GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTCCAAGGCCG ACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG

AGGCGAGTGCTGA (205)

ABLPNB.02 (bispecific antibody containing anti-PD-Ll H12 clone in IgG form and anti-41 BB 41 B01.03 clone in scFv form)

Amino acid Nucleotide sequence sequence (5’—>3’) (Ν’—>С’) (Seq ID (Seq ID No.) No.) Heavy Heavy chain EVQLVESGGGGLVQ GAGGTGCAGCTGGTGGAGA component H12 PGGSLRLSCAASG GCGGAGGAGGACTGGTGCA FTFSSYDMSWVRQ ACCCGGAGGCAGCCTGAGA APGKSLEWVATIS CTGAGCTGCGCTGCCAGCGG DAGGYIYYSDSVK CTTCACCTTCAGCAGCTACG GRFTISRDNAKNS ACATGAGCTGGGTGAGACA LYLQMNSLRDEDT GGCCCCTGGCAAAAGCCTGG AVYICAREFGKRY AG TGGGTGGCCACCATCTCC ALDYWGQGTTVT GATGCGGGCGGCTACATCTA VSSASTKGPSVFPL TTACTCCGACAGCGTGAAGG APSSKSTSGGTAA GCAGGTTCACCATCAGCAGG LGCLVKDYFPEPV GACAACGCCAAGAACAGCC TVSWNSGALTSGV TGTACCTGCAGATGAACAGC HTFPAVLQSSGLY CTGAGGGATGAGGACACCG S LS S VVTVPSSS LG CCGTGTACATCTGCGCCAGG TQTYICNVNHKPS GAGTTCGGCAAAAGGTACG NTKVDKKVEPKSC CCCTGGACTACTGGGGCCAG DKTHTCPPCPAPEL GGCACAACCGTGACCGTGA

LGGPSVFLFPPKPK GCAGCgctAgcAccAAgGGCCC

- 57 045275

DTLM IS RTPE VTC V CTCTGTGTTCCCTCTGGCCCC

VVDVSHEDPEVKF TTCCTCTAAATCCACCTCTG NWYVDGVEVHNA KTKPREEQYASTY RVVSVLTVLHQD WLNGKEYKCKVS NKALPAPIEKTISK AKGQPREPQVYTL PPSREEMTKNQVS LTCLVKGFYPSDIA VEWESNGQPENN YKTTPPVLDSDGS FFLYSKLTVDKSR WQQGNVFSCSVM HEALHNHYTQKSL SLSPGK (206) GCGGAACCGCTGCTCTGGGC TGTCTGGTCAAGGACTACTT CCCTGAGCCCGTGACCGTGT CTTGGAATTCTGGCGCTCTG ACCAGCGGAGTGCACACCTT TCCAGCTGTGCTGCAGTCCT CCGGCCTGTACTCTCTGTCC TCTGTCGTGACAGTGCCTTC CAGCTCTCTGGGCACCCAGA CCTACATCTGCAACGTGAAC CACAAGCCCTCCAACACCAA GGTGGACAAGAAGGTGGAA CCCAAGTCCTGCGACAAGAC CCACACCTGTCCTCCATGTC Linker GGGGSGGGGSGG GGS (207) CTGCTCCAGAACTGCTGGGC GGACCCTCCGTGTTCCTGTT scFv VL QSVLTQPPSASGTP CCCTCCAAAGCCTAAGGACA 41B01 GQRVTISSCSGSSSN CCCTGATGATCTCCCGGACC .03 IGNNYVTWYQQL PGTAPKLLIYADS HRPSGVPDRFSGS KSGTSASLAISGLR SEDEADYYCATW DYSLSGYVFGCGT KLTVL (208) CCTGAAGTGACCTGCGTGGT GGTGGATGTGTCCCACGAGG ATCCAGAAGTGAAGTTCAAT TGGTACGTGGACGGCGTGGA AGTGCACAATGCCAAGCC AAGCCTAGAGAGGAACAGT ACGCCTCCACCTACAGAGTG Linker GGGGSGGGGSGG GGSGGGGS (209) GTGTCCGTGCTGACTGTGCT GCACCAGGATTGGCTGAACG VH EVQLLESGGGLVQ PGGSLRLSCAASG FTFSSYDMSWVRQ APGKCLEWVSWIS YSGGSIYYADSVK GCAAAGAGTACAAGTGCAA GGTGTCCAACAAGGCCCTGC CTGCTCCTATCGAAAAGACC ATCAGCAAGGCCAAGGGCC AGCCTAGGGAACCCCAGGTT TACACCCTGCCTCCAAGCCG

- 58 045275

GRFTISRDNSKNTL YLQMNSLRAEDTA VYYCARDAQRNS MREFDYWGQGTL

VTVSS (210)

GGAAGAGATGACCAAGAAC

CAGGTGTCCCTGACCTGCCT

CGTGAAGGGCTTCTACCCTT

CCGATATCGCCGTGGAATGG

GAGAGCAATGGCCAGCCTG

AGAACAACTACAAGACAAC

CCCTCCTGTGCTGGACTCCG

ACGGCTCATTCTTCCTGTAC

TCCAAGCTGACCGTGGACAA

GTCCAGATGGCAGCAGGGC

AACGTGTTCTCCTGCTCCGT

GATGCACGAGGCCCTGCACA

ATCACTACACCCAGAAGTCC

CTGTCTCTGAGCCCTGGAAA

AGGCGGCGGAGGATCTGGC

GGAGGTGGTAGCGGAGGGCG

GTGGATTCCAGTCTGTTCTG

ACCCAGCCTCCTTCCGCTTC

TGGCACCCCTGGAcAGAGAG

TGACCATCTCTTGCTCCGGC

TCCTCCTCCAACATCGGCAA

CAACTACGTGACCTGGTATC

AGCAGCTGCCCGGCACAGCT

CCCAAACTGCTGATCTACGC

CGACTCTCACAGACCTTCCG

GCGTGCCCGATAGATTCTCC

GGCTCTAAGTCTGGCACCTC

TGCCAGCCTGGCTATCAGCG

GCCTGAGATCTGAGGACGA

GGCCGACTACTACTGCGCCA

CCTGGGATTATTCCCTGTCC

GGCTACGTGTTCGGGCTGCGG

CACAAAACTGACAGTGCTCG

GAGGCGGAGGAAGTGGTGG

CGGAGGTTCAGGTGGTGGTG

- 59 045275

GTAGTGGCGGAGGGCGGATC

AGAAGTTCAGCTGTTGGAGT

CAGGTGGCGGCTTGGTGCAA

CCAGGTGGAAGTCTGAGACT

CAGCTGTGCTGCCAGCGGCT

TTACCTTCAGCTCCTACGAC

ATGAGCTGGGTTCGACAAGC

TCCCGGAAAGTGCTTGGAGT

GGGTTTCCTGGATCTCCTAC

TCCGGCGGCAGCATCTATTA

CGCCGACAGCGTGAAAGGC

CGGTTTACCATCTCTCGGGGA

TAACAGCAAGAATACCCTCT

ACCTCCAATGAACTCTCTG

AGAGCCGAGGACACTGCTGT

GTACTATTGCGCCAGAGATG ccCAGCGGAACTCCATGAGA

GAGTTCGACTACTGGGGACA

AGGCACCCTGGTCACCGTGT

CTAGTTGA (211)

Easy

Light chain Η12 DIQMTQSPSSLSAS

GACATCCAGATGACCCAGA component

VGDRVTITCKASQ

GCCCTAGCAGCCTGAGCGCT

DVTPAVAWYQQK

AGCGTGGGCGACAGGGTGA

PGKAPKLLIYSTSS

CCATCACCTGCAAGGCCAGC

RYTGVPSRFSGSG

CAGGATGTGACCCCTGCCGT

SGTDFTFTISSLQPE

GGCCTGGTACCAGCAGAAG

DIATYYCQQHYTT

CCCGGCAAGGCCCCCAAGCT

PLTFGQGTKLEIK

GCTGATCTACAGCACCAGCA

RTVAAPSVFIFPPS

GCAGGTACACCGGCGTGCCC

DEQLKSGTASVVC

AGCAGGTTTAGCGGAAGCG

LLNNFYPREAKVQ

GCAGCGGCACCGACTTCACC

WKVDNALQSGNS

TTCACCATCAGCAGCCTGCA

QESVTEQDSKDST

GCCCGAGGACATCGCCACCT

YSLSSTLTLSKADY

ACTACTGCCAGCAGCACTAC

EKHKVYACEVTH

ACCACCCCTCTGACCTTCGG

- 60 045275

QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (30) ATCAAGAGAACCGTGGCCG CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTC CAAGGCCG ACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (212)

ABLPNB.03 (bispecific antibody containing anti-PD-LI H12 clone in IgG form and anit-41BB 41B02 clone in scFv form

Amino acid Nucleotide subsequence sequence (5’—>3’) (Ν’—>C’) (Seq ID (Seq ID No.)

No.)

Heavy Heavy chain EVQLVESGGGGLVQ GAGGTGCAGCTGGTGGAGA component H12 PGGSLRLSCAASG GCGGAGGAGGACTGGTGCA FTFSSYDMSWVRQ ACCCGGAGGCAGCCTGAGA APGKSLEWVATIS CTGAGCTGCGCTGCCAGCGG DAGGYIYYSDSVK CTTCACCTTCAGCAGCTACG GRFTISRDNAKNS ACATGAGCTGGGTGAGACA LYLQMNSLRDEDT GGCCCCTGGCAAAAGCCTGG AVYICAREFGKRY AGTGGGTGGCCACCATCTCC ALDYWGQGTTVT GATCGGGCGGCTACATCTA VSSASTKGPSVFPL TTACTCCGACAGCGTGAAGG

- 61 045275

APSSKSTSGGTAA LGCLVKDYFPEPV TVSWNSGALTSGV HTFPAVLQSSGLY SLSSVVTVPSSSLG TQTYICNVNHKPS NTKVDKKVEPKSC DKTHTCPPCPAPEL LGGPSVFLFPPKPK DTLMISRTPEVTCV VVDVSHEDPEVKF NWYVDGVEVHNA KTKPREEQYASTY RVVSVLTVLHQD WLNGKEYKCKVS NKALPAPIE KTISK AKGQPREPQVYTL PPSREEMTKNQVS LTCLVKGFYPSDIA VEWESNGQPENN YKTTPPVLDSDGS FFLYSKLTVDKSR WQQGNVFSCSVM HEALHNHYTQKSL SLSPGK(213) Linker GGGGSGGGGSGG GGS (214) scFv 41B02 VL QSVLTQPPSASGTP GRRVTISSCSGSSSN IGNNYVTWYQQL PGTAPKLLIYADS HRPSGVPDRFSGS KSGTSASLAISGLR SEDEADYYCATW

GCAGGTTCACCATCAGCAGG

GACAACGCCAAGAACAGCC

TGTACCTGCAGATGAACAGC

CTGAGGGATGAGGACACCG

CCGTGTACATCTGCGCCAGG

GAGTTCGGGCAAAAGGTACG

CCCTGGACTACTGGGGCCAG

GGCACAACCGTGACCGTGA

GCAGCgctAgcAccAAgGGCCC

CTCTGTGTTCCCTCTGGCCCC

TTCCTCTAAATCCACCTCTG

GCGGAACCGCTGCTCTGGGC

TGTCTGGTCAAGGACTACTT

CCCTGAGCCCGTGACCGTGT

CTTGGAATTCTGGCGCTCTG

ACCAGCGGAGTGCACACCTT

TCCAGCTGTGCTGCAGTCCT

CCGGCCTGTACTCTCTGTCC

TCTGTCGTGACAGTGCCTTC

CAGCTCTCTGGGCACCCAGA

CCTACATCTGCAACGTGAAC

CACAAGCCCTCCAACACCAA

GGTGGACAAGAAGGTGGAA

CCCAAGTCCTGCGACAAGAC

CCACACCTGTCCTCCATGTC

CTGCTCCAGAACTGCTGGGC

GGACCCTCCGTGTTCCTGTT

CCCTCCAAAGCCTAAGGACA

CCCTGATGATCTCCCGGACC

CCTGAAGTGACCTGCGTGGT

GGTGGATGTGTCCCACGAGG

ATCCCGAAGTGAAGTTCAAT

TGGTACGTGGACGCGTGGA

AGTGCACAACGCCAAGCC

- 62 045275

DYSLSGYVFGCGT KLTVL(215)

Linker GGGGSGGGGSGG

GGSGGGGS (216)

VH EVQLLESGGGLVQ PGGSLRLSCAASG

FTFSGYDMSWVR QAPGKCLEWVSVI YPDDGNTYYADS

VKGRFTISRDNSK NTLYLQMNSLRAE DAAVYYCAKHGG QKPTTKSSSAYG

MDGWGQGTLVTV SS (217)

AAGCCTAGAGAGGAACAGT ACgccTCCACCTACCGGGTGG TGTCCGTGCTGACCGTTCTG CACCAGGATTGGCTGAACGG CAAAGAGTACAAGTGCAAG GTGTCCAACAAGGCCCTGCC TGCCCCTATCGAAAAGACCA TCTCTAAGGCCAAGGGCCAG CCCCGGGAACCTCAAGTGTA CACCTTGCCTCCCAGCCGGG AAGAGATGACCAAGAACCA GGTGTCCC TGACCTGCCTGG TTAAGGGCTTCTACCCCTCC GATATCGCCGTGGAATGGGA GTCTAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCT CCTGTGCTGGACTCCGACGG CTC ATTCTTCCTGT ACTCC A A GCTGACCGTGGACAAGTCTC GGTGGCAGCAGGGCAACGT GTTCTCCTGCTCTGTGATGC ACGAGGCCCTGCACAACCAC TACACCCAGAAGTCCCTGTC CCTGTCTCCCGGCAAAGGTG GGGGGGGATCTGGTGGTGGT GGATCAGGGGGTGGGGGGT CTCAAAGCGTACTCACCCAA CCTCCATCTGCATCCGGTAC ACCTGGTCGGCGAGTAACCA TCTCCTGCTCTGGGAGCTCT TCTAATATTGGTAACAACTA TGTCACCTGGTATCAGCAGT TGCCTGGGACAGCACCCAAA CTTCTTATATATGCCGATAG CCATCGG CCTTCCGGCGTAC

- 63 045275

CCGATCGCTTCTCCGGGTCA AAATCTGGAACATCTGCCTC

ACTCGCAATTAGTGGATTGC GATCTGAGGATGAAGCAGA TTATTATTGCGCTACCTGGG ATTATTCACTTTCTGGCTAC GTCTTTGGTtgtGGAACAAAA CTTACCGTGTTGGGCGGCGG AGGAAGCGGAGGCGGCGGT TCTGGTGGTGGCGGTAGCGG AGGTGGTGGATCTGAGGTTC AACTGTTGGAGTCAGGTGGC GGACTTGTCCAGCCTGGCGG GTCT CTGAGGCTGAGTTGCG CTGCTTCTGGGTTTACTTTTT CAGGATATGACATGAGTTGG GTACGTCAGGCTCCAGGTAA GtgcCTCGAATGGGTCTCCGT TATCTATCCCGATGATGGAA ATACTTACTACGCTGACAGT GTGAAAGGCAGGTTCACAAT CAGTAGGGACAATTCTAAAA ATACACTCTACCTCCAGATG AACTCACTTCGAGCCGAGGA CGCCGCCGTATATT ACTGTG CCAAACACGGCGGGCAAA ACCCACTACTAAATCCAGTA GTGCTTACGGGATGGATGGC TGGGGACAGGGGACATTGG

TCACTGTATCTTCCtga (218)

Light component

Light chain Η12 DIQMTQSPSSLSAS GACATCCAGATGACCCAGA

VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT

DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA

PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC

RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT

- 64 045275

WK VDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCCAGCAGACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (32) ATCAAGAGAACCGTGGCCG

CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTCCAAGGCCG ACTACGAGAAG CACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (219)

ABLPNB.04 (bispecific antibody containing anti-PD-Ll Bb clone in the form of IgG and anti-4-1BB

BO 1 clone in scFv form)

Amino acid Nucleotide sequence sequence (5’—>3’) (Seq ID No.)

- 65 045275

(Ν’—>С’) (Scq ID No.) Heavy Heavy chain EVQLVESGGGGLVQ GAAGTGCAGCTGGTTGAATC component B6 PGGSLRLSCAASG TGGCGGCGGATTGGTTCAGC FTFSSYDMSWVRQ CTGGCGGATCTCTGAGACTG APGKSLEWVATIS TCTTGTGCCGCCTCCGGCTT DAGGYIYYRDSV CACCTTCTCCAGCTACGATA KGRFTISRDNAKN TGTCCTGGGTCCGACAGGCC SLYLQMNSLRDED CCTGGCAAGTCTTTGGAATG T A V Y IC ARELPWR GGTCGCC ACC ATCTCTG ACG YALDY WGQGTTV CTGGCGGCTACATCTACTAC TVSSASTKGPSVFP CGGGACTCTGTGAAGGGCA LAPSS KSTSGGT A G ATTC ACC ATC AGCCGGG AC ALGCLVKDYFPEP AACGCCAAGAACTCCCTGTA VTVSWNSGALTSG CCTGCAGATGAACAGCCTGC VHTFPAVLQSSGL GCGACGAGGATACCGCCGT YSLSSVVTVPSSSL GTACATCTGTGCTAGAGAGC GTQTYICNVNHKP TGCCTTGGAGATACGCCCTG SNTKVDKKVEP KS GATTATTGGGGCCAGGGCAC CDKTHTCPPCPAP CACAGTGACCGTGTCCTCTG ELLGGPSVFLFPPK CTTCTACCAAGGGACCCAGC PKDTLMISRTPEVT GTGTTCCCTCTGGCTCCTTCC CVVVDVSHEDPEV AGCAAGTCTACCTCTGGCGG KFNWYVDGVEVH AACAGCTGCTCTGGGCTGCC NAKTKPREEQYAS TGG TCAAGGACTACTTTCCT TYRVVSVLTVLHQ GAGCCTGTGACAGTGTCCTG DWLNGKEYKCKV GAACTCTGGCGCTCTGACAT SNKALPAPIEKTIS CTGGCGTGCACACCTTTCCA KAKGQPREPQVYT GCAGTGCTGCAGTCCTCCGG LPPSREEMTKNQV CCTGTACTCTCTGTCCTCTGT S LTCLVKGFYPS DI CGTGACCGTGCCTTCC AGCT AVEWESNGQPEN CTCTGGGAACCCAGACCTAC NYKTTPPVLDSDG ATCTGCAATGTGAACCACAA S FFLYS KLTVDKS R GCCTTCC AAC ACC AAGGTGG WQQGNVFSCSVM ACAAGAAGGTGGAACCCAA

- 66 045275

HEALHNHYTQKSL GTCCTGCGACAAGACCCACA SLSPGK (220) CCTGTCCTCCATGTCCTGCTC Linker scFv VL GGGGSGGGGSGG CAGAACTGCTCGGCGGACCT GGS (221) TCCGTGTTCCTGTTTCCTCA QSVLTQPPSASGTP AAGCCTAAGGACACCCTGAT 41В01 Linker VH GRRVTISCSGSSSN GATCTCTCGGACCCCTGAAG IGNNYVTWYQQL TGACCTGCGTGGTGGTGGAT PGTAPKLLIYADS GTGTCCCACGAGGATCCAGA HRPSGVPDRFSGS AGTGAAGTTCAATTGGTACG KSGTSASLAISGLR TGGACGGCGTGGAAGTGCA SEDEADYYCATW CAATGCCAAGACCAAGCCTA DYSLSGYVF GCGT GAGAGGAACAGTACGCCTC KLTVL (222) CACCTACAGAGTGGTGTCCG GGGGSGGGGSGG TGCTGACTGTGCTGCACCAG GGSGGGGS (223) GATTGGCTGAACGGCAAAG EVQLLESGGGLVQ AGTACAAGTGCAAGGTGTCC PGGSLRLSCAASG AACAAGGCCCTGCCTGCTCC FTFSSYDMSWVRQ T ATCGAAAAGACCATСAGCA APGKCLEWVSWIS AGGCCAAGGGCCAGCCTAG YSGGSIYYADSVK GGAACCCCAGGTTT ACACCC GRFTISRDNSKNTL TGCCTCCAAGCCGGGAAGA YLQMNSLRAEDTA GATGACCAAGAACCAGGTG VYYCARDGQRNS TCCCTGACCTGCCTCGTGAA MREFDYWGQGTL GGGCTTCTACCCTTCCGATA VTVSS (224) TCGCCGTGGAATGGGAGAG CAATGGCCAGCTGAGAAC AACTACAAGACAACCCCTCC TGTGCTGGACTCCGACGGCT cattcttcctgtactccaag CTGACCGTGGACAAGTCCAG ATGGCAGCAGGGCAACGTG TTCTCCTGCTCCGTGATGCA CGAGGCCCTGCACAATCACT ACACCCAGAAGTCCCTGTCT CTGAGCCCTGGAAAAGGCG

- 67 045275

GCGGAGGATCTGGCGGAGG

TGGTAGCGGAGGCGGTGGA

TCTCAGTCTGTTCTGACCCA

GCCTCCTTCCGCTTCTGGCA

CCCCTGGAAGAAGAGTGAC

CATCTCTTGCTCCGGCTCCTC

CTCCAACATCGGCAACAACT

ACGTGACCTGGTATCAGCAG

CTGCCCGGCACAGCTCCCAA

ACTGCTGATCTACGCCGACT

CTCACAGACCTTCCGGCGTG

CCCGATAGATTCTCCGGCTC

TAAGTCTGGCACCTCTGCCA

GCCTGGCTATCAGCGGCCTG

AGATTCTGAGGACGAGGCCG

ACTACTACTGCGCCACCTGGG

GATTATTCCCTGTCCGGCTA

CGTGTTCGGCTGCGGCACAA

AACTGACAGTGCTCGGAGGC

GGAGGAAGTGGTGGCGGAG

GTTCAGGTGGTGGTGGTAGT

GGCGGAGGCGGATCAGAAG

TTCAGCTGTTGGAGTCAGGT

GGCGGCTTGGTGCAACCAGG

TGGAAGTCTGAGACTCAGCT

GTGCTGCCAGCGGCTTTACC

TTCAGCTCCTACGACATGAG

CTGGGTTCGACAAGCTCCCG

GAAAGTGCTTGGAGTGGGTT

TCCTGGATCTCCTACTCCGG

CGGCAGCATCTATTACGCCG

ACAGCGTGAAAGGCCGGTTT

ACCATCTCTCGGGATAACAG

CAAGAATACCCTCTACCTCC

AAATGAACTCTCTGAGAGCC

- 68 045275

GAGGACACTGCTGTGTACTA TTGCGCCAGAGATGGCCAGC GGAACTCCATGAGAGAGTTC GACTACTGGGGACAAGGCA CCCTGGTCACCGTGTCTAGT Lightweight Light chain Wb component DIQMTQSPSSLSAS VGDRVT1TCKASQ TGA (225) GACATCCAGATGACCCAGA GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQGTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCC AGCAGC ACT AC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (34) ATCAAGAGAACCGTGGCCG

CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGC A AC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC

CCTGACCCTGTCCAAGGCCG ACTACGAGAAGCACAAGGT

- 69 045275

GTACGCCTGCGAAGTGACCC

ACCAGGGACTGTCTAGCCCC

GTGACCAAGTCCTTCAACAG

AGGCGAGTGCTGA (226)

ABLPNB.05 (bispecific antibody containing anti-PD-Ll Bb clone in IgG form and anti-4-1 B B

VO 1.01 clone in scFv form)

Amino acid Nucleotide sequence sequence (5’—>3’) (Ν’—>С’) (Seq ID (Seq ID No.) No.) Heavy Heavy chain EVQLVESGGGGLVQ GAAGTGCAGCTGGTTGAATC component Wb PGGSLRLSCAASG TGGCGGCGGATTGGTTCAGC FTFSSYDMSWVRQ CTGGCGGATCTCTGAGACTG APGKSLEWVATIS TCTTGTGCCGCCTCCGGCTT DAGGYIYYRDSV CACCTTCTCCAGCTACGATA KGRFTISRDNAKN TGTCCTGGGTCCGACAGGCC SLYLQMNSLRDED CCTGGCAAGTCTTTGGAATG TAVYICARELPWR GGTCGCCACCATCTCTGACG YALDYWGQGTTV CTGGCGGCTACATCTACTAC TVSSASTKGPSVFP CGGGACTCTGTGAAGGGCA LAPSSKSTSGGTA GATTCACCATCAGCCGGGAC ALGCLVKDYFPEP AACGCCAAGAACTCCCTGTA VTVSWNS GALTS G CCTGCAGATGAACAGCCTGC VHTFPAVLQSSGL GCGACGAGGATACCGCCGT YSLS SVVTVPSSSL GTACATCTGTGCTAGAGAGC GTQTYICNVNHKP TGCCTTGGAGATACGCCCTG SNTKVDKKVEPKS GATTATTGGGGCCAGGGCAC CDKTHTCPPCPAP CACAGTGACCGTGTCCTCTG ELLGGPSVFLFPPK CTTCTACCAAGGGACCCAGC PKDTLMISRTPEVT GTGTTCCCTCTGGCTCCTTCC CVVVDVSHEDPEV AGCAAGTCTACCTCTGGCGG KFNWYVDGVEVH AACAGCTGCTCTGGGCTGCC NAKTKPREEQYAS TGGTCAAGGACTACTTTCCT TYRVVSVLTVLHQ GAGCCTGTGACAGTGTCCTG

- 70 045275

DWLNGKEYKCKV SNKALPAPIEKTIS GAACTCTGGCGCTCTGACAT CTGGCGTGCACACCTTTCCA Linker KAKGQPREPQVYT LPPSREEMTKNQV SLTCLVKGFYPSDI AVEWESNGQPEN NYKTTPPVLDSDG SFFLYSKLTVDKSR WQQGNVFSCSVM HEALHNHYTQKSL SLSPGK (227) GGGGSGGGGSGG GCAGTGCTGCAGTCCTCCGG CCTGTACTCTCTGTCCTCTGT CGTGACCGTGCCTTCCAGCT CTCTGGGAACCCAGACCTAC ATCTGCAATGTGAACCACAA GCCTTCCAACACCAAGGTGG ACAAGAAGGTGGAACCCAA GTCCTGCGACAAGACCCACA CCTGTCCTCCATGTCCTGCTC CAGAACTGCTCGGCGGACCT scFv GGS (228) VL QSVLTQPPSASGTP TCCGTGTTCCTGTTTCCTCA AAGCCTAAGGACACCCTGAT 41B01 GRRVTISSCSGSSSN GATCTCTCGGACCCCTGAAG .01 IGNNYVTWYQQL TGACCTGCGTGGTGGTGGAT PGTAPKLLIYADS HRPSGVPDRFSGS KSGTSASLAISGLR SEDEADYYCATW DYSLSGYVFGCGT KLTVL (229) Linker GGGGSGGGGSGG GTGTCCCACGAGGATCCAGA AGTGAAGTTCAATTGGTACG TGGACGGCGTGGAAGTGCA CAATGCCAAGACCAAGCCTA GAGAGGAACAGTACGCCTC CACCTACAGAGTGGTGTCCG TGCTGACTGTGCTGCACCCAG GGSGGGGS (230) VH EVQLLESGGGLVQ GATTGGCTGAACGGCAAAG AGTACAAGTGCAAGGTGTCC PGGSLRLSCAASG FTFSSYDMSWVRQ APGKCLEWVSWIS YSGGSIYYADSVK GRFTISRDNSKNTL YLQMNSLRAEDTA VYYCARDAQRNS MREFDYWGQGTL VTVSS (231) AACAAGGCCCTGCCTGCTCC TATCGAAAAGACCATCAGCA AGGCCAAGGGCCAGCCTAG GGAACCCCAGGTTTACACCC TGCCTCCAAGCCGGGAAGA GATGACCAAGAACCAGGTG TCCCTGACCTGCCTCGTGAA GGGCTTCTACCCTTCCGATA TCGCCGTGGAATGGGAGAG CAATGGCCAGCCTGAGAAC

- 71 045275

AACTACAAGACAACCCCTCC

TGTGCTGGACTCCGACGGCT

CATTCTTCCTGTACTCCAAG

CTGACCGTGGACAAGTCCAG

ATGGCAGCAGGGCAACGTG

TTCTCCTGCTCCGTGATGCA

CGAGGCCCTGCACAATCACT

ACCACCAGAAGTCCCTGTCT

CTGAGCCCTGGAAAAGGCG

GCGGAGGATCTGGCGGAGG

TGGTAGCGGAGGCGGTGGA

TCTCAGTCTGTTCTGACCCA

GCCTCCTTCCGCTTCTGGCA

CCCCTGGAAGAAGAGTGAC

CATCTCTTGCTCCGGCTCCTC

CTCCAACATCGGCAACAACT

ACGTGACCTGGTATCAGCAG

CTGCCCGGCACAGCTCCCAA

ACTGCTGATCTACGCCGACT

CTCACAGACCTTCCGGCGTG

CCCGATAGATTCTCCGGCTC

TAAGTCTGGCACCTCTGCCA

GCCTGGCTATCAGCGGCCTG

AGATTCTGAGGACGAGGCCG

ACTACTACTGCGCCACCTGGG

GATTATTCCCTGTCCGGCTA

CGTGTTCGGCTGCGGCACAA

AACTGACAGTGCTCGGAGGC

GGAGGAAGTGGTGGCGGAG

GTTCAGGTGGTGGTGGTAGT

GGCGGAGGCGGATCAGAAG

TTCAGCTGTTGGAGTCAGGT

GGCGGCTTGGTGCAACCAGG

TGGAAGTCTGAGACTCAGCT

GTGCTGCCAGCGGCTTTACC

- 72 045275

Light component

Light chain Wb

TTCAGCTCCTACGACATGAG

CTGGGTTCGACAAGCTCCCG

GAAAGTGCTTGGAGTGGGTT

TCCTGGATCTCCTACTCCGG

CGGCAGCATCTATTACGCCG

ACAGCGTGAAAGGCCGGTTT

ACCATCTCTCGGGATAACAG

CAAGAATACCCTCTACCTCC

AAATGAACTCTCTGAGAGCC

GAGGACACTGCTGTGTACTA

TTGCGCCAGAGATGccCAGC

GGAACTCCATGAGAGAGTTC

GACTACTGGGGACAAGGCA

CCCTGGTCACCGTGTCTAGT

TGA (232)

DIQMTQSPSSLSAS GACATCCAGATGACCCAGA

VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQ GTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACT ACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (36) ATCAAGAGAACCGTGGCCG

CTCCCTCCGTGTTCATCTTCC

CACCATCTGACGAGCAGCTG

AAGTCCGGCACCGCTTCTGT

- 73 045275

CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTCCAAGGCCG ACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCT TCAACAG AGGCGAGTGCTGA (233)

ABLPNB.06 (bispecific antibody containing anti-PD-Ll Bb clone in IgG form and anti-4-1 B B

VO 1.02 clone in scFv form)

Amino acid Nucleotide subsequence sequence (5’—>3’) (N'^C') (Seq ID (Seq ID No.)

No.)

Heavy Heavy chain EVQLVESGGGGLVQ GTGCAGCTGGTTGAATCTGG component B6 PGGSLRLSCAASG CGGCGGATTGGTTCAGCCTG FTFSSYDMSWVRQ GCGGATCTCTGAGACTGTCT APGKSLEWVATIS TGTGCCGCCTCCGGCTTCAC DAGGYIYYRDSV CTTCTCCAGCTACGATATGT KGRFTISRDNAKN CCTGGGTCCGACAGGCCCCT SLYLQMNSLRDED GGCAAGTCTTTGGAATGGGT TAVYICARELPWR CGCCACCATCTCTGACGCTG YALDYWGQGTTV GCGGCTACATCTACTACCGG TVSSASTKGPSVFP GACTCTGTGAAGGGCAGATT LAPSSKSTSGGTA CACCATCAGCCGGGGACAAC ALGCLVKDYFPEP GCCAAGAACTCCCTGTACCT VTVSWNSGALTSG GCAGATGAACAGCCTGCGC VHTFPAVLQSSGL GACGAGGATACCGCCGTGTA YSLSSVVTVPSSSL CATCTGTGCTAGAGAGCTGC

- 74 045275

GTQTYICNVNHKP SNTKVDKKVEPKS CTTGGAGATACGCCCTGGAT TATTGGGGCCAGGGCACCAC Linker scFv VL CDKTHTCPPCPAP ELLGGPSVFLFPPK PKDTLMISRTPEVT CVVVDVSHEDPEV KFNWYVDGVEVH NAKTKPREEQYAS TYRVVSVLTVLHQ DWLNGKEYKCKV SNKALPAPIEKTIS KAKGQPREPQVYT LPPSREEMTKNQV SLTCLVKGFYPSDI AVEWESNGQPEN NYK TTPPVLDSDG SFFLYSKLTVDKSR WQQGNVFSCSVM HEALHNHYTQKSL SLSPGK (234) GGGGSGGGGSGG GGS (235) QSVLTQPPSASGTP AGTGACCGTGTCCTCTGCTT CTACCAAGGGACCCAGCGTG TTCCCTCTGGCTCCTTCCAGC AAGTCTACCCTTGGCGGAAC AGCTGCTCTGGGCTGCCTGG TCAAGGACTACTTTCCTGAG CCTGTGACAGTGTCCTGGAA CTCTGGCGCTCTGACATCTG GCGTGCACACCTTTCCAGCA GTGCTGCAGTCCTCCGGCCT GTACTCTCTGTCCTCTGTCGT GACCGTGCCTTCCAGCTCTC TGGGAACCCAGACCTACATC TGCAATGTGAACCACAAGCC TTCCAACACCAAGGTGGACA AGAAGGTGGAACCCCAAGTC CTGCGACAAGACCCACACCT GTCCTCCATGTCCTGCTCCA GAACTGCTCGGCGGACCTTC CGTGTTCCTGTTTCCTCCAA AGCCTAAGGACACCCTGATG 41В01 .02 Linker GRRVTISSCSGSSSN IGNNYVTWYQQL PGTAPKLLIYADS HRPSGVPDRFSGS KSGTSASLAISGLR SEDEADYYCATW DYSLSGYVFGCGT KLTVL (236) GGGGSGGGGSGG GGSGGGGS (237) ATCTCTCGGACCCCTGAAGT GACCTGCGTGGTGGTGGATG TGTCCCACGAGGATCCAGAA GTGAAGTTCAATTGGTACGT GGACGGCGTGGAAGTGCAC AATGCCAAGACCAAGCCTA GAGAGGAACAGTACGCCTC CACCTACAGAGTGGTGTCCG TGCTGACTGTGCTGCACCCAG GATTGGCTGAACGGCAAAG

- 75 045275

VH EVQLLESGGGLVQ AGTACAAGTGCAAGGTGTCC

PGGSLRLSCAASG AACAAGGCCCTGCCTGCTCC FTFSSYDMSWVRQ TATCGAAAAGACCATCAGCA APGKCLEWVSWIS AGGCCAAGGGCCAGCCTAG

YSGGSIYYADSVK GGAACCCCAGGTTTACACCC

GRFTISRDNSKNTL TGCCTCCAAGCCGGGAAGA

YLQMNSLRAEDTA GATGACCAAGAACCAGGTG

VYYCARDAQRQS TCCCTGACCTGCCTCGTGAA MREFDYWGQGTL GGGCTTCTACCCTTCCGATA

VTVSS (238) TCGCCGTGGAATGGGAGAG

CAATGGCCAGCCTGAGAAC

AACTACAAGACAACCCCTCC

TGTGCTGGACTCCGACGGCT CATTCTTCCTGTACTCCAAG CTGACCGTGGACAAGTCCAG ATGGCAGCAGGGCAACGTG TTCTCCTGCTCCGTGATGCA CGAGGCCCTGCACAATCACT ACACCCAGAAGTCCCTGTCT CTGAGCCCTGGAAAAGGCG GCGGAGGATCTGGCGGAGG TGGTAGCGGAGGCGGTGGA TCTCAGTCTGTTCTGACCCA GCCTCCTTCCG CTTCTGGC A CCCCTGGAAGAAGAGTGAC CATCTCTTGCTCCGGCTCCTC CTCCAACATCGGCAACAACT ACGTGACCTGGTATCAGCAG CTGCCCGGCACAGCTCCCAA ACTGCTGATCTACGCCGACT CTCACAGACCTTCCGGCGTG CCCGATAGATTCTCCGGCTC TAAGTCTGGCACCTCTGCCA GCCTGGCTATCAGCGGCCTG AGATCTGAGGACGAGGCCG

- 76 045275

ACTACTACTGCGCCACCTGGG GATTATTCCCTGTCCGGCTA CGTGTTCGGCTGCGGCACAA AACTGACAGTGCTCGGAGGC GGAGGAAGTGGTGGCGGAG GTTCAGGTGGTGGTGGTAGT GGCGGAGGCGGATCAGAAG TTCAGCTGTTGGAGTCAGGT GGCGGCTTGGTGCAACCAGG TGGAAGTCTGAGACTCAGCT GTGCTGCCAGCGGCTTTACC TTCAGCTCCTACGACATGAG CTGGGTTCGACAAGCTCCCG GAAAGTGCTTGGAGTGGGTT TCCTGGATCTCCTACTCCGG CGGCAGCATCTATTACGCCG ACAGCGTGAAAGGCCGGTTT ACCATCTCTCGGGATAACAG CAAGAATACCCTCTACCTCC AAATGAACTCTCTGAGAGCC GAGGACACTGCTGTGTACTA TTGCGCCAGAGATGccCAGC GGCAATCCATGAGAGAGTTC GACTACTGGGGACAAGGCA CCCTGGTCACCGTGTCTAGT TGA (239) Lightweight Light chain Wb DIQMTQSPSSLSAS GACATCCAGATGACCCAGA component VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQGTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC

- 77 045275

DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG

LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (38) ATC AAGAGAACCGTGGCCG

CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTCCAAGGCCG ACTACGAGAAG CACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (240)

ABLPNB.07 (bispecific antibody containing anti-PD-Ll Bb clone in the form of IgG and anti-4-1 B 41B01.03 clone in the form of scFv)

Heavy Heavy chain Amino acid Nucleotide sequence sequence (5’—>3’) (Ν’—>С’) (Scq ID (Scq ID No.) No.) EVQLVESGGGLVQ GAAGTGCAGCTGGTTGAATC Wb component PGGSLRLSCAASG TGGCGGCGGATTGGTTCAGC FTFSSYDMSWVRQ CTGGCGGATCTCTGAGACTG APGKSLEWVATIS TCTTGTGCCGCCTCCGGGCTT

- 78 045275

DAGGYIYYRDSV CACCTTCTCCAGCTACGATA

A ACGCCAAGAACTCCCTGTA VTVSWNSGALTSG CCTGCAGATGAACAGCCTGC VHTFPAVLQSSGL GCGACGAGGATACCGCCGT YSLSSVVTVPSSSL GTACATCTGTGCTAGAGAGC GTQTYICNVNH KP TGCCTTGGAGATACGCCCTG SNTKVDKKVEPKS GATTATTGGGGCCAGGGCAC CDKTHTCPPCPAP CACAGTGACCGTGTCCTCTG ELLGGPSVFLFPPK CTTCTACCAAGGGACCCAGC PKDTLMISRTPEVT GTGTTCCCTCTGGCTCCTTCC CVVVDVSHEDPEV AGCAAGTCTACCTCTGGCGG KFNWYVDGVEVH AACAGCTGCTCTGGGCTGCC NAKTKPREEQYAS TGGTCAAGGACTACTTTCCT TYRVVSVLTVLHQ GAGCCTGTGACAGTGTCCTG DWLNGKEYKCKV GAACTCTGGCGCTCTGACAT SNKALPAPIEKTIS CTGGCGTGCACACCTTTCCA KAKGQPREPQVYT GCAGTGCTGCAGTCCTCCGG LPPSREEMTKNQV CCTGTACTCTCTGTCCTCTGT

S LTCLVKGFYPS DI CGTGACCGTGCCTTCC AGCT AVEWESNGQPEN CTCTGGGAACCCAGACCTAC NYKTTPPVLDSDG ATCTGCAATGTGAACCACAA SFFLYSKLTVDKSR GCCTTCCAACACCAAGGTGG WQQGNVFSCSVM ACAAGAAGGTGGAACCCAA HEALHNHYTQKSL GTCCTGCGACAAGACCCACA SLSPGK (241)CCTGTCCTCCATGTCCTGCTC

GGGGSGGGGSGG

CAGAACTGCTCGGCGGACCT

GGS (242)

TCCGTGTTCCTGTTTCCTCA

VL QSVLTQPPSASGTP AAGCCTAAGGACACCCTGAT

GQRVTISSCSGSSSN GATCTCTCGGACCCCTGAAG

- 79 045275

scFv IGNNYVTWYQQL TGACCTGCGTGGTGGTGGAT 41B01 PGTAPKLLIYADS GTGTCCCACGAGGATCCAGA .03 HRPSGVPDRFSGS AGTGAAGTTCAATTGGTACG KSGTSASLAISGLR TGGACGGCGTGGAAGTGCA SEDEADYYCATW CAATGCCAAGACCAAGCCTA DYSLSGYVFGCGT GAGAGGAACAGTACGCCTC KLTVL (243) CACCTACAGAGTGGTGTCCG ; Linker GGGGSGGGGSGG TGCTGACTGTGCTGCACCCAG GGSGGGGS (244) GATTGGCTGAACGGCAAAG VH EVQLLESGGGLVQ AGTACAAGTGCAAGGTGTCC PGGSLRLSCAASG AACAAGGCCCTGCCTGCTCC FTFSSYDMSWVRQ TATCGAAAAGACCATCAGCA APGKCLEWVSWIS AGGCCAAGGGCCAGCCTAG YSGGSIYYADSVK GGAACCCCAGGTTTACACCC GRFTISRDNSKNTL TGCCTCCAAGCCGGGAAGA YLQMNSLRAEDTA GATGACCAAGAACCAGGTG VYYCARDAQRNS TCCCTGACCTGCCTCGTGAA MREFDYWGQGTL GGGCTTCTACCCTTCCGATA VTVSS (245) TCGCCGTGGAATGGGAGAG CAATGGCCAGCCTGAGAAC AACTACAAGACAACCCCTCC TGTGCTGGACTCCGACGGCT CATTCTTCCTGTACTCCAAG CTGACCGTGGACAAGTCCAG ATGGCAGCAGGGCAACGTG TTCTCCTGCTCCGTGATGCA CGAGGCCCTGCACAATCACT ACACCCAGAAGTCCCTGTCT CTGAGCCCTGGAAAAGGCG GCGGAGGATCT GGCGGAGG TGGTAGCGGAGGCGGTGGA TCTCAGTCTGTTCTGACCCA GCCTCCTTCCGCTTCTGGCA CCCCTGGAcAGAGAGTGACC ATCTCTTGCTCCGGCTCCTCC

- 80 045275

TCCAACATCGGCA AC A ACTA

CGTGACCTGGTATCAGCAGC

TGCCCGGCACAGCTCCCAAA

CTGCTGATCTACGCCGACTC

TCACAGACCTTCCGGCGTGC

CCGATAGATTCTCCGGCTCT

AAGTCTGGCACCTCTGCCAG

CCTGGCTATCAGCGGCCTGA

GATCTGAGGACGAGGCCGA

CTACTACTGCGCCACCTGGG

ATTATTCCCTGTCCGGCTAC

GTGTTCGGCTGCGGCACAAA

ACTGACAGTGCTCGGAGGCG

GAGGAAGTGGTGGCGGGAGG

TTCAGGTGGTGGTGGTAGTG

GCGGAGGCGGATCAGAAGT

TCAGCTGTTGGAGTCAGGTG

GCGGCTTGGTGCAACCAGGT

GGAAGTCTGAGACTCAGCTG

TGCTGCCAGCGGCTTTACCT

TCAGCTCCTACGACATGAGC

TGGGTTCGACAAGCTCCCGG

AAAGTGCTTGGAGTGGGTTT

CCTGGATCTCCTACTCCGGC

GGCAGCATCTATTACGCCGA

CAGCGTGAAAGGCCGGTTTA

CCATCTCTCGGGATAACAGC

AAGAATACCCTTCTACCTCCA

AATGAACTCTCTGAGAGCCG

AGGACACTGCTGTGTACTAT

TGCGCCAGAGATGccCAGCG

GAACTCCATGAGAGAGTTCG

ACTACTGGGGACAAGGCAC

CCTGGTCACCGTGTCTAGTT

GA (246)

- 81 045275

Lightweight Light chain Wb DIQMTQSPSSLSAS GACATCCAGATGACCCAGA component VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQGTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTS ACC ATS AGC AGCCTGC A QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (40) ATCAAGAGAACCGTGGCCG CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTCCAAGGCCG ACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (247)

ABLPNB.08

- 82 045275 (bispecific antibody containing anti-PD-Ll Bb clone in the form of IgG and anti-4-1 B B 41 BO 1.04 clone in the form of scFv)

Amino acid subsequence Nucleotide sequence (5’—>3’) (Ν’—>С’) (Scq ID (Scq ID No.) No.) Heavy Heavy chain EVQLVESGGGGLVQ GAAGTGCAGCTGTTGAATC Wb component PGGSLRLSCAASG TGGCGGCGGATTGGTTCAGC

FTFSSYDMSWVRQ CTGGCGGATCTCTGAGACTG APGKSLEWVATIS TCTTGTGCCGCCTCCGGGCTT DAGGYIYYRDSV CACCTTCTCCAGCTACGATA KGRFTISRDNAKN TGTCCTGGGTCCGACAGGCC SLYLQMNSLRDED CCTGGCAAGTCTTTGGAATG TAVYICARELPWR GGTCGCCACCATCTCTGACG YALDYWGQGTTV CTGGCGGCTACATCTACTAC TVSSASTKGPSVFP CGGGACTCTGTGAAGGGCA LAPSSKSTSGGTA GATTCACCATCAGCCGGGAC ALGCLVKDYFPEP AACGCCAAGAACTCCCTGTA VTVSWNSGALTSG CCTGCAGATGAACAGCCTGC VHTFPAVLQSSGL GCGACGAGGATACCGCCGT YSLSSVVTVPSSSL GTACATCTGTGCTAGAGAGGC GTQTYICNVNHKP TGCCTTGGAGATACGCCCTG SNTKVDKKVEPKS GATTATTGGGGCCAGGGCAC CDKTHTCPPCPAP CACAGTGACCGTGTCCTCTG ELLGGPSVFLFPPK CTTCTACCAAGGGACCCAGC PKDTLMISRTPEVT GTGTTCCCTCTGGCTCCTTCC CVVVDVSHEDPEV AGCAAGTCTACCTCTGGCGG KFNWYVDGVEVH AACAGCTGCTCTGGGCTGCC NAKTKPREEQYAS TGGTCAAGGACTACTTTCCT TYRVVSVLTVLHQ GAGCCTGTGACAGTGTCCTG DWLNGKEYKCKV GAACTCTGGCGCTCTGACAT SNKALPAPIEKTIS CTGGCGTGCACACCTTTCCA KAKGQPREPQVYT GCAGTGCTGCAGTCCTCCGG LPPSREEMTKNQV CCTGTACTCTCTGTCCTCTGT SLTCLVKGFYPSDI CGTGACCGTGCCTTCCAGCT

- 83 045275

AVEWESNGQPEN NYKTTPPPVLDSDG SFFLYSKLTVDKSR CTCTGGGAACCCAGACCTAC ATCTGCAATGTGAACCACAA GCCTTCCAACACCAAGGTGG WQQGNVFSCSVM ACAAGAAGGTGGAACCCAA HEALHNHYTQKSL GTCCTGCGACAAGACCCACA SLSPGK (248) CCTGTCCTCCATGTCCTGCTC Linker GGGGSGGGGSGG CAGAACTGCTCGGCGGACCT GGS (249) TCCGTGTTCCTGTTTCCTCA scFv VL QSVLTQPPSASGTP AAGCCTAAGGACACCCTGAT 41 BOI GQRVTISSCSGSSSN GATCTCTCGGACCCCTGAAG .04 IGNNYVTWYQQL TGACCTGCGTGGTGGTGGAT PGTAPKLLIYADS GTGTCCCACGAGGATCCAGA HRPSGVPDRFSGS AGTGAAGTTCAATTGGTACG KSGTSASLAISGLR TGGACGGCGTGGAAGTGCA SEDEADYYCATW CAATGCCAAGACCAAGCCTA DYSLSGYVFGCGT GAGAGGAACAGTACGCCTC KLTVL (250) CACCTACAGAGTGGTGTCCG Linker GGGGSGGGGSGG TGCTGACTGTGCTGCACCCAG GGSGGGGS (251) GATTGGCTGAACGGCAAAG VH EVQLLESGGGLVQ AGTACAAGTGCAAGGTGTCC PGGSLRLSCAASG AACAAGGCCCTGCCTGCTCC FTFSSYDMSWVRQ TATCGAAAAGACCATCAGCA APGKCLEWVSWIS AGGCCAAGGGCCAGCCTAG YSGGSIYYADSVK GGAACCCCAGGTTTACACCC GRFTISRDNSKNTL TGCCTCCAAGCCGGGAAGA YLQMNSLRAEDTA GATGACCAAGAACCAGGTG VYYCARDAQRQS TCCCTGACCTGCCTCGTGAA MREFDYWGQGTL GGGCTTCTACCCTTCCGATA VTVSS (252) TCGCCGTGGAATGGGAGAG

CAATGGCCAGCCTGAGAAC

AACTACAAGACAACCCCTCC

TGTGCTGGACTCCGACGGCT

CATTCTTCCTGTACTCCAAG

CTGACCGTGGACAAGTCCAG

ATGGCAGCAGGGCAACGTG

- 84 045275

TTCTCCTGCTCCGTGATGCA

CGAGGCCCTGCACAATCACT ACACCCAGAAGTCCCTGTCT CTGAGCCCTGGAAAAGGCG GCGGAGGATCTGGCGGAGG TGGTAGCGGAGGCGGTGGA TCTCAGTCTGTTCTGACCCA GCCTCCTTCCGCTTCTGGCA CCCCTGGAcAGAGAGTGACC ATCTCTTGCTCCGGCTCCTCC TCCAACATCGGCAACAACTA CGTGACCTGGTATCAGCAGC TGCCCGGCACA GCTCCCAAA CTGCTGATCTACGCCGACTC TCACAGACCTTCCGGCGTGC CCGATAGATTCTCCGGCTCT AAGTCTGGCACCTCTGCCAG CCTGGCTATCAGCGGCCTGA GATCTGAGGACGAGGCCGA CTACTACTGCGCCACCTGGG ATTATTCCCTGTCCGGCTAC GTGTTCGGCTGCGGCACAAA ACTGACAGTGCTCGGAGGCG GAGGAAGTGGTGGCGGAGG TTCAGG TGGTGGTGGTAGTG GCGGAGGCGGATCAGAAGT TCAGCTGTTGGAGTCAGGTG GCGGCTTGGTGCAACCAGGT GGAAGTCTGAGACTCAGCTG TGCTGCCAGCGGCTTTACCT TCAGCTCCTACGACATGAGC TGGGTTCGACAAGCTCCCGG AAAGTGCTTGGAGTGGGTTT CCTGGATCTCCTACTCCGGC GGCAGCATCTATTACGCCGA

- 85 045275

CAGCGTGAAAGGCCGGTTTA CCATCTCTCGGGATAACAGC AAGAATACCCTTCTACCTCCA AATGAACTCTCTGAGAGCCG AGGACACTGCTGTGTACTAT TGCGCCAGAGATGccCAGCG GCAATCCATGAGAGAGTTCG ACTACTGGGGACAAGGCAC CCTGGTCACCGTGTCTAGTT GA (253) Lightweight Light chain Wb DIQMTQSPSSLSAS GACATCCAGATGACCCAGA component VGDRVTITCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQGTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG QGLSSPVTKSFNR CCAGGGCACCAAGCTGGAG GEC (42) ATCAAGAGAACCGTGGCCG CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA

- 86 045275

GCAGGACTCCAAGGACAGC

ACCTACTCCCTGTCCTCTAC

CCTGACCCTGTCCAAGGCCG ACTAGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (254) ABLPNB.09

(bispecific antibody containing anti-PD-Ll Bb clone in the form of IgG and anti-4-1 B B 41 B02 clone in the form of scFv)

Amino acid Nucleotide sequence sequence (5’—>3’) (N’^C’) (Seq ID (Seq ID No.) No.) Heavy Heavy chain EVQLVESGGGGLVQ GAAGTGCAGCTGGTTGAATC component Wb PGGSLRLSCAASG TGGCGGCGGATTGGTTCAGC FTFSSYDMSWVRQ CTGGCGGATCTCTGAGACTG APGKSLEWVATIS TCTTGTGCCGCCTCCGGCTT DAGGYIYYRDSV CACCTTCTCCAGCTACGATA KGRFTISRDNAKN TGTCCTGGGTCCGACAGGCC SLYLQMNSLRDED CCTGGCAAGTCTTTGGAATG TAVYICARELPWR GGTCGCCACCATCTCTGACG YALDYWGQGTTV CTGGCGGCTACATCTACTAC TVSSASTKGPSVFP CGGGACTCTGTGAAGGGCA LAPSSKSTSGGTA GATTCACCATCAGCCGGGAC ALGCLVKDYFPEP AAC GCCAAGAACTCCCTGTA VTVSWNSGALTSG CCTGCAGATGAACAGCCTGC VHTFPAVLQSSGL GCGACGAGGATACCGCCGT YSLSSVVTVPSSSL GTACATCTGTGCTAGAGAGC GTQTYICNVNHKP TGCCTTGGAGATACGCCCTG SNTKVDKKVEPKS GATTATTGGGGCCAGGGCAC CDKTHTCPPCPAP CACAGTGACCGTGTCCTCTG ELLGGPSVFLFPPK CTTCTACCAAGGGACCCAGC PKDTLMISRTPEVT GTGTTCCCTCTGGCTCCTTCC

- 87 045275

CVVVDVSHEDPEV AGCAAGTCTACCTCTGGCGG KFNWYVDGVEVH AACAGCTGCTCTGGGCTGCC NAKTKPREEQYAS TGGTCAAGGACTACTTTCCT TYRVVSVLTVLHQ GAGCCTGTGACAGTGTCCTG DWLNGKEYKCKV GAACTCTGGCGCTCTGACAT SNKALPAPIEKTIS CTGGCGTGCACACCTTTCCA KAKGQPREPQVYT GCAGTGCTGCAGTCCTCCGG LPPSREEMTKNQV CCTGTACTCTCTGTCCTCTGT SLTCLVKGFYPSDI CGTGACCGTGCCTTCCAGCT AVEWESNGQPEN CTCTGGGCACCCAGACCTAC NYKTTPPPVLDSDG ATCTGCAACGTGAACCACAA SFFLYSKLTVDKSR GCCCTCCAACACCAAGGTGG WQQGNVFSCSVM ACAAGAAGGTGGAACCCAA HEALHNHYTQKSL GTCCTGCGACAAGACCCACA SLSPGK (255) CCTGTCCTCCATGTCCTGCTC Linker GGGGSGGGGSGG CAGAACTGCTGGGCGGACCC GGS (256) TCCGTGTTCCTGTTCCCCTCCA scFv VL QSVLTQPPSASGTP A AGCCTA AGGACACCTGAT 41B02 GRRVTISSCSGSSSN GATCTCCCGGACCCCTGAAG IGNNYVTWYQQL TGACCTGCGTGGTGGTGGAT PGTAPKLLIYADS GTGTCCCACGAGGATCCCCGA HRPSGVPDRFSGS AGTGAAGTTCAATTGGTACG KSGTSASLAISGLR TGGACGGCGTGGAAGTGCA SEDEADYYCATW CAACGCCAAGACCAAGCCT DYSLSGYVFGCGT AGAGAGGAACAGTACgccTC KLTVL (257) CACCTACCGGGTGGTGTCCG Linker GGGGSGGGGSGG TGCTGACCGTTCTGCACCAG GGSGGGGS (258) GATTGGCTGAACGGCAAAG VH EVQLLESGGGLVQ AGTACAAGTGCAAGGTGTCC PGGSLRLSCAASG AACAAGGCCCTGCCTGCCCC FTFSGYDMSWVR TATCGAAAAGACCATCTCTTA QAPGKCLEWVSVI AGGCCAAGGGCCAGCCCCG YPDDGNTYYADS GGAACCTCAAGTGTACACCT VKGRFTISRDNSK TGCCTCCCAGCCGGGAAGAG

ATGACCAAGAACCAGGTGTC

- 88 045275

NTLYLQMNSLRAE DAAVYYCAKHGG QKPTTKSSSAYG MDGWGQGTLVTV SS (259)

CCTGACCTGCCTGGTTAAGG GCTTCTACCCCTCCGATATC

GCCGTGGAATGGGAGTCTAA CGGCCAGCCCGAGAACAAC TACAAGACCACCCCTCCTGT GCTGGACTCCGACGGCTCAT TCTTCCTGTACTCCAAGCTG ACCGTGGACAAGTCTCGGTG GCAGCAGGGCAACGTGTTCT CCTGCTCTGTGATGCACGAG GCCCTGCACAACCACTACAC CCAGAAGTCCCTGTCCCTGT CTCCCGGCAAAGGTGGGGG GGGATCTGG TGGTGGTGGAT CAGGGGGTGGGGGGTCTCA AAGCGTACTCACCCAACCTC CATCTGCATCCGGTACACCT GGTCGGCGAGTAACCATCTC CTGCTCTGGGAGCTCTTCTA ATATTGGTAACAACTATGTC ACCTGGTATCAGCAGTTGCC TGGGACAGCACCCAAACTTC TTATATATGCCGATAGCCAT CGGCCTTCCGGCGTACCCGA TCGCTTCTCCGGGTCAAA AT CTGGAACATCTGCCTCACTC GCAATTAGTGGATTGCGATC TGAGGATGAAGCAGATTATT ATTGCGCTACCTGGGATTAT TCACTTTCTGGCTACGTCTTT GGTtgtGGAACAAAACTTACC GTGTTGGGCGGCGGAGGAA GCGGAGGCGGCGGTTCTGGT GGTGGCGGTAGCGGAGGTG

GTGGATCTGAGGTTCAACTG

- 89 045275

TTGGAGTCAGGTGGCGGACT TGTCCAGCCTGGCGGGTCTC TGAGGCTGAGTTGCGCTGCT TCTGGGTTTACTTTTTCAGG ATATGACATGAGTTGGGTAC GTCAGGCTCCAGGTAAGtgcC TCGAATGGGTCTCCGTTATC TATCCCGATGATGGAAATAC TTACTACGCTGACAGTGTGA AAGGCAGGTTCACAATCAGT AGGGACAATTCTAAAAATA C ACTCTACCTCCAGATGAACT CACTTCGAGCCGAGGACGCC GCCGTATATTACTGTGCCAA ACACGGCGGGCAAAAACCC ACTACTAAATCCAGTAGTGC TTACGGGATGGATGGCTGGG GACAGGGGACATTGGTCACT GTATCTTCCtga (260) Lightweight Light chain Wb DIQMTQSPSSLSAS GACATCCAGATGACCCAGA component VGDRVT1TCKASQ GCCCTAGCAGCCTGAGCGCT DVTPAVAWYQQK AGCGTGGGCGACAGGGTGA PGKAPKLLIYSTSS CCATCACCTGCAAGGCCAGC RYTGVPSRFSGSG CAGGATGTGACCCCTGCCGT SGTDFTFTISSLQPE GGCCTGGTACCAGCAGAAG DIATYYCQQHYTT CCCGGCAAGGCCCCCAAGCT PLTFGQGTKLEIK GCTGATCTACAGCACCAGCA RTVAAPSVFIFPPS GCAGGTACACCGGCGTGCCC DEQLKSGTASVVC AGCAGGTTTAGCGGAAGCG LLNNFYPREAKVQ GCAGCGGCACCGACTTCACC WKVDNALQSGNS TTCACCATCAGCAGCCTGCA QESVTEQDSKDST GCCCGAGGACATCGCCACCT YSLSSTLTLSKADY ACTACTGCCAGCAGCACTAC EKHKVYACEVTH ACCACCCCTCTGACCTTCGG CCAGGGCACCAAGCTGGAG

- 90 045275

QGLSSPVTKSFNR

GEC (44)

ATCAAGAGAACCGTGGCCG CTCCCTCCGTGTTCATCTTCC CACCATCTGACGAGCAGCTG AAGTCCGGCACCGCTTCTGT CGTGTGCCTGCTGAACAACT TCTACCCTCGGGAAGCCAAG GTGCAGTGGAAGGTGGACA ATGCCCTGCAGTCCGGCAAC TCCCAAGAGTCTGTGACCGA GCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTAC CCTGACCCTGTC CAAGGCCG ACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCC ACCAGGGACTGTCTAGCCCC GTGACCAAGTCCTTCAACAG AGGCGAGTGCTGA (261)

The constructed vectors were transiently expressed in ExpiCHO-S™ cells (Thermo Fisher, A29127) using a kit (ExpiFectamine™ CHO Kit, Thermo, A29129), cultured in ExpiCHO™ expression medium (Thermo, A29100-01) under conditions from 30 to 37 °C for 7-15 days in a CO ₂ incubator equipped with a rotating shaker. Plasmid DNA (250 μg) and ExpiFectamin CHO reagent (800 μl) were mixed with Opti-MEM® I medium (final volume 20 ml) and left at room temperature for 5 min. The mixed solution was added to 6x10 ⁶ ExpiCHO cells cultured in ExpiCHO expression medium and mixed gently in an incubator shaker at 37°C in a humidified atmosphere with 8% CO ₂ in the air. 18 h after transfection, 1.5 ml ExpiFectamin CHO Transfection Enhancer 1 and 60 ml ExpiFectamin CHO Transfection Feed were added to each flask.

Each BsAb was purified from cell culture supernatant using recombinant protein A affinity chromatography (Hitrap Mabselect Sure, GE Healthcare, 28-4082-55) and gel filtration chromatography on a HiLoad 26/200 Superdex200 prep grade column (GE Healthcare, 28-55). 9893-36). SDS-PAGE (NuPage 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent, 1200 series) with a SE-HPLC column (SWXL SE-HPLC column, TOSOH, G3000SWXL) were performed to detect and confirm the size and purity of each BsAb. Purified proteins were concentrated in PBS by ultrafiltration using an Amicon Ultra 15 30K device (Merck, UFC903096), and protein concentrations were assessed using nanodroplets (Thermo, Nanodrop One). When using a two-vector system, the ratio between light and heavy chains can be from 1:1 to 1:3 by weight. Alternatively, a single-vector system can also be used, which contains both circuits in one single vector.

The resulting anti-PD-L1/anti-4-1BB bispecific antibodies are named H12x41B01(ABLPNB.01), H12x41B01.03(ABLPNB.02), H12x41B02(ABLPNB.03), B6x41B01(ABLPNB.04), B6x41B01.01(ABLPNB .05), B6x41B01.01(ABLPNB.06), B6x41B01.03(ABLPNB.07), B6x41B01.04(ABLPNB.08) and B6x41B02(ABLPNB.09), respectively, where the first refers to the clone in the form of IgG, and the last refers to the clone in scFv form.

Example 4. Characterization of bispecific antibodies PD-L1x4-1BB 4.1. Binding of bispecific antibodies.

To evaluate the PD-L1 and 4-1BB binding activity of the bispecific antibodies prepared in Example 3, the BsAbs (ABLPNB.01, ABLPNB.03, ABLPNB.04 and ABLPNB.07) were subjected to a DACE (Dual Antigen Capture ELISA) assay. Briefly, microtiter plates were coated with 100 ng/well human PD-L1-Fc protein (Sinobio, 10084-H02H) in PBS at 4°C overnight, then blocked with 100 μl/well 1% BSA for 2 h at 37°C. Threefold dilutions of each BsAb, starting at 100 nM, were added to each well and incubated for 2 h at 37°C. The plates were washed with PBS/Tween and then incubated with 50 ng/well human 4-lBB-His protein (Sinobio, 16498-H08H) in 1% BSA for 1 h at 37°C. The plates were washed with PBS/Tween and then incubated with Anti-His HRP (Roche, catalog number: 11965085001) for 1 h at 37°C. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-650 nm. The results are shown in Fig. 7A and 7B. As shown in FIG. 7A and 7B, all BsAbs tested can bind to both human PD-L1 and human 4-1BB proteins with high potency.

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4.2 Serum stability of bispecific antibody.

To evaluate the serum stability of bispecific antibodies against PD-L1 and 4-1BB (ABLPNB.05, ABLPNB.06, ABLPNB.07 and ABLPNB.08), BsAb was incubated in human serum for 3.7, 14 days at 37°C. Binding activity was analyzed using the DACE (Dual Antigen Capture ELISA) assay. Briefly, microtiter plates were coated with 100 ng/well human PD-L1-Fc protein (Sinobio, 10084-H02H) in PBS at 4°C overnight, then blocked with 100 μl/well 1% BSA for 2 h at 37°C. . Threefold dilutions of each BsAb, starting at 100 nM, were added to each well and incubated for 2 h at 37°C. The plates were washed with PBS/Tween and then incubated with 50 ng/well human 4-1BB-His protein (Sinobio, 16498-H08H) in 1% BSA for 1 h at 37°C. The plates were washed with PBS/Tween and then incubated with Anti-His HRP (Roche, catalog number: 11965085001) for 1 h at 37°C. After washing, the plates were developed with TMB substrate and analyzed on a spectrophotometer at OD 450-650 nm. The binding activity of each BsAb (ABLPNB.05 - ABLPNB.08) to both antigens was comparable for each sample. This means that BsAbs are stable in human serum for 2 weeks at 37°C. Representative data is shown in FIG. 8.

4.3. Possibility of developing bispecific antibodies.

The development feasibility of the physicochemical properties of PD-L1 and 4-1BB BsAb (ABLPNB.02 and ABLPNB.07) was assessed. The quality attributes of bispecific antibodies (BsAbs) were assessed by several analytical methods. Briefly, purity was measured using size exclusion high-performance liquid chromatography (SE-HPLC), and both BsAbs showed high purity of more than 99%. Thermal stability was analyzed by fluorescently tagged protein thermal shift (PTS) using real-time polymerase chain reaction (RT-PCR). Their melting point exceeded 67°C, indicating the stable structural integrity of the studied substances. To assess the solubility of molecules, proteins were concentrated to 20 mg/ml using ultrafiltration (Amicon Ultra-15 centrifugal concentrator). As a result, no visible particles were observed by visual inspection, and no increase in aggregates was confirmed by SE-HPLC. The isoelectric point (pI) of ABLPNB.02 and ABLPNB.07 measured by capillary isoelectric focusing (cIEF) was 8.26 and 8.35, respectively. This pI range is suitable for downstream processing and formulation development. In general, as shown in table. 19, testing has shown that the BsAbs tested (ABLPNB.02 and ABLPNB.07) have the appropriate physicochemical properties for successful development.

Table 19

Characteristic Way ABLPNB.07 ABLPNB.02 Purity SEC 99.6 99.8 Thermal stability PTS 67.0 67.1 76.8 80.5 Solubility Visual inspection Easily concentrated to 20 mg/ml, transparent Easy to concentrate to 20 mg/ml, transparent pi cIEF 8.26 8.35

4.4. The activity of bispecific antibodies stimulates the 4-1BB signal.

The 4-1BB cell assay was used to test the ability of the bispecific antibodies (ABLPNB.01, ABLPNB.03, ABLPNB.07, and ABLPNB.08) to stimulate the 4-1BB signal. This assay used the GloResponse™ NFkB-1uc2/4-1BB Jurkat cell line (Promega, cat. no. CS196004) as effector cells, and a cancer cell line expressing or not expressing PD-L1 was used as target cells. The GloResponse™ NFkB-1uc2/4-1BB Jurkat cell line is genetically modified to stably express 4-1BB and luciferase downstream of the response element. Luciferase expression is induced by antibody binding to the 4-1BB receptor. Briefly, plate HCC1954 (PD-L1 expression) or BT474 or NCI-N87 (no PD-L1 expression) at 2.5 x 104 cells per well on a white 96-well assay plate in 100 µl culture medium (RPMI1640+10% FBS). Cultivated overnight in an incubator humidified at 37°C + 5% CO2. After overnight cultivation, 100 μl of culture medium was removed and 25 μl of assay medium (RPMI1640+1% FBS) was added to the pre-plated target cells. 25 µl of bispecific antibodies (starting from 15 nM diluted 8 times or starting from 1.5 nM diluted 4 times) and BMUR or monoclonal antibody 41B01 (starting from 20 nM diluted 10 times or starting from 133 nM diluted 6 times) added to the tablet. The harvested GloResponse™ NFkB-1uc2/4-1BB Jurkat cell line was resuspended in assay media. Added 25 µl of cell

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Claims (6)

GloResponse™ NFkB-1uc2/4-1BB Jurkat line per well, yielding 2.5x104 cells per well on the plate. Cultivated for 6 hours at 37°C + 5% CO2 in a humidified incubator. During the incubation time, Bio-Glo™ reducing reagent was added according to the manufacturer's instructions. After 6 hours of incubation, 75 per well of Bio-Glo™ reagent was added to the assay plate. Wait 5 min and analyze the luminescence using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software. The results are shown in Fig. 9A-9F. As shown in FIG. 9A, 9B, monoclonal antibody against 4-1BB showed very limited activation of the 4-1BB signal compared to BUMR. 41B01 (assayed in Fc-crosslinked form) showed only weak activation of the 4-1BB signal (Fig. 9B). In the case of the bispecific antibody cocultured with PD-L1-negative cancer cells (Figures 9C and 9D), the bispecific antibody PD-L1x4-1BB showed no activation of the 4-1BB signal (Figure 9C) and a weak activation of the 4-1BB signal at Fc cross-linking, as shown in FIG. 9C and 9D respectively. In the presence of PD-L1, i.e. when co-cultured with PD-L1-positive target cells (Figures 9E and 9F), the bispecific antibody PD-L1x4-1BB showed activation of the 4-1BB signal, which was dependent on the presence of tumor antigen (PD-L1). 1. Anti-PD-L1/anti-4-1BB bispecific antibody containing an αnmu-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, where the anti-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 2, CDR3 VH having the amino acid sequence SEQ ID NO: 4, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; and the anti-4-1BB antibody or antigen binding fragment thereof comprises a CDR1 VH having the amino acid sequence of SEQ ID NO: 10, a CDR2 VH having the amino acid sequence of SEQ ID NO: 12, a CDR3 VH having the amino acid sequence of SEQ ID NO: 14, - 93 045275 CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20. 2. Anti-PD-L1/anti-4-1BB bispecific antibody containing an anti-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, where αntu-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 2, CDR3 VH having the amino acid sequence SEQ ID NO: 4, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; and the anti-4-1BB antibody or antigen binding fragment thereof comprises CDR1 VH having the amino acid sequence of SEQ ID NO: 10, CDR2 VH having the amino acid sequence of SEQ ID NO: 12, CDR3 VH having the amino acid sequence of SEQ ID NO: 16, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20. 3. Anti-PD-L1/anti-4-1BB bispecific antibody containing an αntu-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, where anti-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 2, CDR3 VH having the amino acid sequence SEQ ID NO: 4, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; and the anti-4-1BB antibody or antigen binding fragment thereof comprises CDR1 VH having the amino acid sequence of SEQ ID NO: 11, CDR2 VH having the amino acid sequence of SEQ ID NO: 13, CDR3 VH having the amino acid sequence of SEQ ID NO: 15, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20. 4. Anti-PD-L1/anti-4-1BB bispecific antibody comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, wherein the anti-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 3, CDR3 VH having the amino acid sequence SEQ ID NO: 5, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; and the anti-4-1BB antibody or antigen binding fragment thereof comprises CDR1 VH having the amino acid sequence of SEQ ID NO: 10, CDR2 VH having the amino acid sequence of SEQ ID NO: 12, CDR3 VH having the amino acid sequence of SEQ ID NO: 14, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20. 4.6. Tumor growth inhibition by bispecific antibodies (in vivo assay). Humanized mice expressing the extracellular domain of human 4-1BB were used. Murine colon adenocarcinoma cells (MC38) were engineered to express human PD-L1. Humanized mice (h4-1BB) were subcutaneously implanted with MC38-hPD-L1 cells. Mice were intraperitoneally injected with Q3D 5 times (five injections of antibody every three days) with the following antibodies: isotype control (10 mg/kg), anti-PD-L1 antibody (H12, 10 mg/kg), anti-4-1BB antibody (41B01 , 10 mg/kg), combination of anti-PD-L1 (H12, 10 mg/kg) and anti-4-1BB (41B01, 10 mg/kg) antibodies and bispecific antibody PD-L1x4-1BB (BsAb) {BLPNB. 01, 13.3 mg/kg}. The volume of tumors was monitored using calipers twice a week during the experiment. The inhibition of tumor growth induced by ABLPNB.01 was significantly greater than that observed for the combination of each targeting monoclonal antibody (see Fig. 11). The scope of the present disclosure is not to be limited by the specific embodiments described, which are intended as separate illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are included within the scope of the present disclosure. Those skilled in the art will appreciate that various modifications and changes may be made to the methods and compositions of the present invention without departing from the spirit or scope of the present disclosure. Thus, the present disclosure is intended to cover modifications and variations of the present invention provided they come within the scope of the appended claims and their equivalents. All publications and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference. CLAIM 4.5. The activity of bispecific antibodies to stimulate the T-cell immune response in humans. To test the ability of the bispecific antibodies to stimulate a human PBMC response, a cytokine production assay was used. Human PBMCs stimulated with human anti-CD3 antibody were used as effector cells. HCC1954 cells, which express PD-L1, were used as target cells. In this system, PBMC (3x10 ⁴ ) were co-cultured with HCC1954 (1x10 ⁴ ) in the presence of human anti-CD3 antibody. Bispecific antibodies (ABLPNB.01 and ABLPNB.04) (starting from 20 nM (=4 μg/ml), diluted in 10 doses) and their analogous monoclonal antibodies (41B01 and H12) (starting from 26.67 nM (=4 μg /ml), diluted into 10 doses) were added to the mixed culture. As shown in FIG. 10A and 10B, only bispecific antibodies can activate T cells in the presence of tumor cells expressing PD-L1 (see FIG. 10A). Moreover, bispecific antibodies activated T cells in a dose-dependent manner (see Fig. 10B). 5. Anti-PD-L1/anti-4-1BB bispecific antibody comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, wherein the anti-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 3, CDR3 VH having the amino acid sequence SEQ ID NO: 5, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; and the anti-4-1BB antibody or antigen binding fragment thereof comprises CDR1 VH having the amino acid sequence of SEQ ID NO: 10, CDR2 VH having the amino acid sequence of SEQ ID NO: 12, CDR3 VH having the amino acid sequence of SEQ ID NO: 16, CDR1 VL having the amino acid sequence of SEQ ID NO: 18, CDR2 VL having the amino acid sequence of SEQ ID NO: 19, and CDR3 VL having the amino acid sequence of SEQ ID NO: 20. 6. Anti-PD-L1/anti-4-1BB bispecific antibody comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof and an anti-4-1BB antibody or an antigen-binding fragment thereof, wherein the anti-PD-L1 antibody or an antigen-binding fragment thereof contains CDR1 VH having the amino acid sequence SEQ ID NO: 1, CDR2 VH having the amino acid sequence SEQ ID NO: 3, CDR3 VH having the amino acid sequence SEQ ID NO: 5, CDR1 VL having the amino acid sequence SEQ ID NO: 6, CDR2 VL having the amino acid sequence of SEQ ID NO: 7, and CDR3 VL having the amino acid sequence of SEQ ID NO: 8; And -