Proteins containing the Kallikrein-related Peptidase 2 antigen-binding domain and their use

Abstract

Embodiments of the present invention provide isolated proteins comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), including monospecific and bispecific antibodies. Additional embodiments of the present invention provide polynucleotides, vectors, host cells encoding hK2-specific proteins, and methods of making and using the same.

Description

[Technical Field]

[0001] (Sequence Listing) This application includes a sequence listing that has been electronically filed in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy was created on 23 June 2020, named JBI6125USNP1_SL.txt, and has a size of 764,265 bytes.

[0002] (Field of invention) The present invention provides an antigen-binding domain that binds to an hK2 protein, which includes an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), a polynucleotide encoding the same, a vector, a host cell, and a method for producing and using the same.

[0003] (background) Prostate cancer is the second most common cancer, the sixth leading cause of cancer death in men, and accounts for 14% (903,500) of all new cancer cases and 6% (258,400) of all cancer deaths in men worldwide. The progression from diagnosis to death of prostate cancer is best classified into a series of clinical stages based on the severity of the disease, hormonal status, and the presence or absence of detectable metastasis: localized disease, elevated prostate-specific antigen (PSA) levels without detectable metastasis after radiotherapy or surgery, and clinical metastasis in the uncastrated or castrated stage. Surgery, radiation therapy, or a combination of both can be curative for patients with localized disease, but a significant proportion of these patients have recurrent disease, evidenced by elevated PSA levels, which can lead to the development of metastasis, particularly in high-risk groups, i.e., progression to the terminal stage of the disease.

[0004] Androgen depletion therapy (ADT) is the standard treatment, and the generally predictable outcomes are a decrease in PSA, a stable period with no tumor growth, followed by an increase in PSA and regrowth as a castration-resistant disease. For many years, ADT has been the standard treatment for patients with metastatic prostate cancer.

[0005] Kallikrein-related peptidase 2 (hK2, HK2) is a trypsin-like enzyme specifically expressed in prostate tissue and prostate cancer, driven by the androgen receptor (AR). hK2 is activated by the transmembrane protease serine 2 (TMPRSS2) and secreted into the prostatic ducts, where it initiates a cascade that enhances sperm motility by cleaving semenogenin, an extracellular matrix in ejaculate. While hK2 expression is limited to prostate and prostate cancer tissue, it has recently been demonstrated that hK2 is detectable in breast cancer strains and primary patient samples after the AR pathway has been appropriately activated by steroid hormones (US Patent Application Publication 2018 / 0326102). Similar to PSA, retrograde release of catalytically inactive hK2 into the bloodstream occurs when the highly structured tissue of the prostate is impaired during hypertrophy or malignant transformation.

[0006] Next-generation hK2-binding domains are needed for therapeutic and diagnostic purposes.

[0007] (overview) This disclosure provides an isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the following: Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163; or HC of the VH of SEQ ID NO: 164 DR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205.

[0008] The disclosure also provides an isolated antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the heavy chain variable region (VH) of SEQ ID NO: 75 and the light chain variable region (VL) of SEQ ID NO: 74.

[0009] This disclosure also provides isolated antigen-binding domains that bind to hK2, including specific VH and VL amino acid sequences.

[0010] This disclosure also provides isolated multispecific proteins comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises: heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity-determining regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138; or HCDR1, HCDR2, and HCD of the VH of SEQ ID NO: 162 R3, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 163; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205. In certain embodiments, the Disclosure provides an isolated multispecific protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162, and the light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0011] The disclosure also provides an isolated multispecific protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the heavy chain variable region (VH) of SEQ ID NO: 75 and the light chain variable region (VL) of SEQ ID NO: 74.

[0012] This disclosure also provides isolated, multispecific proteins comprising an antigen-binding domain that binds to hK2 containing specific VH and VL amino acid sequences.

[0013] This disclosure also provides isolated chimeric antigen receptors (CARs) comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2).

[0014] This disclosure also provides an isolated chimeric antigen receptor (CAR) comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises: heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 163; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205. In certain embodiments, the Disclosure provides an isolated chimeric antigen receptor (CAR) comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162, and the light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0015] The disclosure also provides an isolated chimeric antigen receptor (CAR) comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the heavy chain variable region (VH) of SEQ ID NO: 75 and the light chain variable region (VL) of SEQ ID NO: 74.

[0016] This disclosure also provides an isolated, multispecific protein comprising a first antigen-binding domain that binds to hK2 and a second antigen-binding domain that binds to a lymphocyte antigen (such as CD3).

[0017] In certain embodiments, the disclosure provides an isolated multispecific protein comprising a first antigen-binding domain that binds to hK2 and a second antigen-binding domain that binds to a lymphocyte antigen, and which is an isolated anti-hK2 / anti-CD3 protein.

[0018] This disclosure also provides an immunoconjugate comprising an isolated antigen-binding domain that binds to hK2 of this disclosure.

[0019] This disclosure also provides an immunoconjugate comprising an isolated protein containing an antigen-binding domain that binds to hK2 of this disclosure.

[0020] The disclosure also provides an immunoconjugate comprising an isolated multispecific protein containing an antigen-binding domain that binds to hK2 of the disclosure.

[0021] This disclosure also provides an immunoconjugate comprising an isolated CAR containing an antigen-binding domain that binds to hK2 of this disclosure.

[0022] This disclosure also provides a pharmaceutical composition comprising an isolated antigen-binding domain that binds to the hK2 of this disclosure.

[0023] The Disclosure also provides a pharmaceutical composition comprising an isolated protein containing an antigen-binding domain that binds to hK2 of the Disclosure.

[0024] The Disclosure also provides a pharmaceutical composition comprising an isolated multispecific protein containing an antigen-binding domain that binds to hK2 of the Disclosure.

[0025] This disclosure also provides pharmaceutical compositions comprising the isolated multispecific proteins of this disclosure.

[0026] The Disclosure also provides a pharmaceutical composition comprising an isolated CAR containing an antigen-binding domain that binds to the hK2 of the Disclosure.

[0027] This disclosure also provides isolated polynucleotides encoding an isolated antigen-binding domain that binds to hK2 of this disclosure.

[0028] This disclosure also provides isolated polynucleotides encoding isolated proteins that include an antigen-binding domain that binds to hK2 of this disclosure.

[0029] The disclosure also provides isolated polynucleotides encoding isolated multispecific proteins that include an antigen-binding domain that binds to hK2 of the disclosure.

[0030] The Disclosure also provides isolated polynucleotides encoding isolated CARs that include an antigen-binding domain that binds to hK2 of the Disclosure.

[0031] This disclosure also provides vectors comprising the polynucleotides of this disclosure.

[0032] Furthermore, the present invention provides a host cell comprising the polynucleotide or vector of the present disclosure.

[0033] The Disclosure also provides a method for treating hK2-expressing cancer in a subject, comprising administering a therapeutically effective amount of an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to hK2, a multispecific protein comprising the antigen-binding domain that binds to hK2, a CAR-T protein comprising the antigen-binding domain that binds to hK2, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat the hK2-expressing cancer.

[0034] The Disclosure also provides a method for reducing the amount of hK2-expressing tumor cells in a subject, comprising administering to the subject for a period of time sufficient to reduce the amount of hK2-expressing tumor cells, an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to hK2, a multispecific protein comprising the antigen-binding domain that binds to hK2, a CAR-T protein comprising the antigen-binding domain that binds to hK2, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0035] The Disclosure also provides a method for preventing the establishment of hK2-expressing cancer in a subject, comprising administering to a subject in need of such treatment an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to said hK2, a multispecific protein comprising the antigen-binding domain that binds to said hK2, a CAR-T protein comprising the antigen-binding domain that binds to said hK2, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure, to prevent the establishment of hK2-expressing cancer in said subject.

[0036] The Disclosure also provides a method for treating a non-cancerous condition in a subject at risk of developing an hK2-expressing cancerous condition, comprising administering to a subject in need of such treatment the non-cancerous condition an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to said hK2, a multispecific protein comprising the antigen-binding domain that binds to said hK2, a CAR-T protein comprising the antigen-binding domain that binds to said hK2, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0037] The Disclosure also provides a method for treating prostate cancer in a subject, comprising administering a therapeutically effective amount of an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to said hK2, a multispecific protein comprising the antigen-binding domain that binds to said hK2, a CAR-T protein comprising the antigen-binding domain that binds to said hK2, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat the prostate cancer.

[0038] The Disclosure also provides a method for treating breast cancer in a subject, comprising administering a therapeutically effective amount of an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to said hK2, a multispecific protein comprising the antigen-binding domain that binds to said hK2, a CAR-T protein comprising the antigen-binding domain that binds to said hK2, an immune conjugate of the Disclosure, or a pharmaceutical composition of the Disclosure to a subject in need for a period of time sufficient to treat said breast cancer.

[0039] The Disclosure also provides a method for detecting prostate cancer or breast cancer in a subject, comprising administering the immunoconjugate of the Disclosure to the subject and detecting the binding of the immunoconjugate to hK2, thereby detecting prostate cancer or breast cancer.

[0040] The Disclosure also provides a kit comprising an antigen-binding domain that binds to hK2, a protein comprising the antigen-binding domain that binds to said hK2, a multispecific protein comprising the antigen-binding domain that binds to said hK2, a CAR-T protein comprising the antigen-binding domain that binds to said hK2, an immunoconjugate of the Disclosure, or a pharmaceutical composition of the Disclosure.

[0041] This disclosure also provides an anti-idiotype antibody that binds to an antigen-binding domain that binds to hK2 of this disclosure.

[0042] The disclosure also provides a chimeric antigen receptor (CAR) comprising an extracellular domain including an antigen-binding domain that binds to hK2, a transmembrane domain, and an intracellular signaling domain optionally including at least one costimulatory domain. [Brief explanation of the drawing]

[0043] The foregoing will become clear from a more detailed description of the exemplary embodiments shown in the accompanying drawings below. TM: Transmembrane. [Figure 1] The sequence alignments of the VH domains of mu11B6 (sequence number 125), hu11B6 (sequence number 5), KL2B357 (sequence number 159), KL2B358 (sequence number 161), KL2B359 (sequence number 139), KL2B360 (sequence number 159), HCF3 (sequence number 6), and HCG5 (sequence number 4) are shown. [Figure 2] The sequence alignments of the VL domains of mu11B6 (sequence number 124), hu11B6 (sequence number 2), KL2B357 (sequence number 160), KL2B358 (sequence number 140), KL2B359 (sequence number 140), KL2B360 (sequence number 140), LDC6 (sequence number 1), and LCB7 (sequence number 3) are shown. [Figure 3] This figure shows the binding epitopes of selected hK2 antibodies mapped onto the hK2 antigen sequence. In this figure, each sequence is disclosed as sequence number 467. [Figure 4A]This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (Figure 1B), and the mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 4B] This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (Figure 1B), and the mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 5] This shows the binding of the anti-CD3 scFv variant expressed in Escherichia coli (E. coli) to CD3. [Figure 6] The alignment of the VL regions of CD3B815 (sequence number 249), CD3W244 (sequence number 250), CD3W245 (sequence number 251), CD3W246 (sequence number 252), CD3W247 (sequence number 253), and CD3W248 (sequence number 254) is shown. [Figure 7] The graph shows the results of hydrogen-deuterium exchange mass spectrometry (HDX-MS) of CD3W245 (CD3ε:CD3W245) bound to human CD3ε and OKT3 (CD3ε:OKT) bound to CD3ε. The amino acid sequences shown represent residues 2-93 of the 105 residues in the ECD domain of CD3ε, corresponding to residues 1-91 of CD3ε SEQ ID NO: 340. A single underline indicates a segment in which the deuteration level was reduced by 10%-30% in the presence of the antibody compared to CD3ε alone, and a double underline indicates a segment in which the deuteration level was reduced by >30%. [Figure 8A] This shows the in vitro target cell cytotoxicity of the KL2B×CD3 bispecific molecule, measured in real time by the incuCyte imaging system, for quantifying target cell death. [Figure 8B] This shows the in vitro target cytotoxicity of the KL2B×CD3 bispecific molecule, as measured by the fluorescent caspase 3 / 7 reagent for measuring apoptosis signals from target cell death. [Figure 9A] This demonstrates in vitro T cell activation and proliferation by a KLK2 × CD3 bispecific antibody, by showing the frequency of CD25-positive cells at different doses. [Figure 9B] This demonstrates in vitro T cell activation and proliferation by a KLK2 × CD3 bispecific antibody by showing the frequency of cells entering the proliferation gate. [Figure 10A] This demonstrates in vitro T-cell INF-γ release induced by a KLK2 x CD3 bispecific antibody. [Figure 10B] This demonstrates in vitro T-cell TNF-α release induced by a KLK2 x CD3 bispecific antibody. [Figure 11] The blueprint for the hK2-binding chimeric artificial receptor (CAR) is shown. hK2-binding scFv was cloned into various CARs in either VH-VL or VL-VH orientation. [Figure 12A] This shows hK2 CAR expression on the surface of T cells. Primary human T cells were electroporated with either no mRNA (mock), or 10 μg of mRNA expressing either the hK2 scFv CAR or an unrelated control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (upper figure), or PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (lower figure), or PE conjugated with biotinylated hK2 (1 μg / mL) and streptavidin. [Figure 12B] This shows hK2 CAR expression on the surface of T cells. Primary human T cells were electroporated with either no mRNA (mock), or 10 μg of mRNA expressing either the hK2 scFv CAR or an unrelated control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (upper figure), or PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (lower figure), or PE conjugated with biotinylated hK2 (1 μg / mL) and streptavidin. [Figure 13]This study demonstrates the cytotoxicity of hK2-positive (VCaP, top figure) and hK2-negative (DU145, bottom figure) tumor cells by hK2 CAR-T cells in a 20-hour flow-based assay at a specified effector-to-target cell (E / T) ratio. Target cells were labeled with Cell Trace Violet (CTV) fluorescent dye 24 hours after transient transfection and then co-cultured with hK2 CAR-T cells. Mock T cells served as a negative effector control. Killing percentage was measured as the ratio of the absolute number of viable (vitality dye-negative) target (CTV-positive) cells remaining in the co-culture to the number of viable targets cultured without CAR-T cells. [Figure 14-1] Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 14-2]Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 14-3] Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-1]This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-2] This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-3]This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 16] This shows interferon-gamma (IFN-γ) production by antigen-stimulated hK2 CAR-T cells. Supernatants were collected from xCELligence-based killing assays after co-culture for approximately 70 hours (VCAP#5E4, DU145#5E3). hK2 CAR-LH and control CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCAP cells, but not during co-culture with hK2-negative DU145 cells. Mean IFN-γ concentration ± standard deviation (pg / mL) from duplicate cultures is shown. [Figure 17-1] As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 17-2]As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 17-3] As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 18] This shows the binding between CAR1-10 constructs and their allogeneic cell antigens (hK2 on target cells), as detected by luciferase expression in JNL cells upon binding. Transduced JNL cells and untransduced JNL cells (UTD) containing the specified CAR constructs (CAR1-10) were co-cultured with target cell lines (VCaP or DU145 cells), and luciferase activity was measured as luminescence intensity. If the luminescence intensity was 1.5 times higher than that of UTD cells in the presence of antigen-expressing cells, the construct was considered active. [Figure 19-1] The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 19-2]The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 19-3] The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 20] This shows the percentage of tumor cell growth inhibition in hK2-positive VCaP cells at effector:target ratios of 1:1 or 0.5:1 by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 21] This shows the percentage of tumor cell growth inhibition in PC3 cells at an effector:target ratio of 1:1 by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 22-1]This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 22-2] This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 22-3] This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 23]This shows IFN-γ release by hK2 CAR-T cells. Supernatant was collected from hK2 CAR-T cells co-cultured overnight (approximately 20 hours) with VCaP and DU145 (5E4 cells) cells in a 1:1 E / T ratio. hK2 CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCaP cells, but not during co-culture with hK2-negative DU145 cells. The mean IFN-γ concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. [Figure 24A-1] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-2] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-3] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-4] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-5]The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-6] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-1] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-2] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-3] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-4] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-5]The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-6] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-1] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-2] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-3] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-1] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-2]The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-3] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-4] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 26] This study shows that hK2 CAR-T cells proliferated more robustly than CD3 / 28 bead-positive controls after co-culture with VCaP cells for 5 days. T cells genetically engineered with various CAR constructs exhibited different proliferative activity and different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell count + / - SEM from three technical replicas. [Figure 27-1] This shows the surface expression of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL), and CAR20 (HK2B359_LH) on the surface of primary T cells. The numbers in each histogram represent the percentage of cells expressing the specified CAR. [Figure 27-2] This shows the surface expression of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL), and CAR20 (HK2B359_LH) on the surface of primary T cells. The numbers in each histogram represent the percentage of cells expressing the specified CAR. [Figure 28-1]This shows the surface expression of CAR17(KL2B413_HL), CAR18(KL2B413_LH), CAR19(KL2B359_HL), and CAR20(KL2B359_LH) on the surface of JNL cells. [Figure 28-2] This shows the surface expression of CAR17(KL2B413_HL), CAR18(KL2B413_LH), CAR19(KL2B359_HL), and CAR20(KL2B359_LH) on the surface of JNL cells. [Figure 29] The figure shows the relative light units (RLU) resulting from luciferase expression in JNL cells mediated by the binding of test CAR-T to its allogeneic receptor in various target cells as specified in the figure. JNL cells containing the specified CAR clones and untransduced NJL cells (UTD) were co-cultured with target cell lines (VCaP, LNCaP / hK2, LNCaP, C4-2B, 22Rv1, or DU145 cells), and luciferase activity was measured as luminescence intensity (RLU, relative light units). KL2B413_HL:CAR17, KL2B413_LH:CAR18, HL2B359_HL:CAR19, KL2B359_LH:CAR20. [Figure 30-1] The figures show the percentage of tumor cell growth inhibition of hK2-positive VCaP cells by transduced CAR-T cells using CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 30-2] The figures show the percentage of tumor cell growth inhibition of hK2-positive VCaP cells by transduced CAR-T cells using CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 31] The figures show the percentage of tumor cell growth inhibition in hK2-negative DU145 cells by transduced CAR-T cells of CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 32] The figures show IFN-γ production by transduced CAR-T cells or untransduced T cells (UTDs) in co-cultures with the cells specified in the figure: CAR17 (KLK2B413HL in the figure), CAR18 (KLK2B413LH in the figure), CAR19 (KLK2B359HL in the figure), and CAR20 (KLK2B359LH in the figure). [Figure 33A-1] Co-culturing transduced CAR-T cells (CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure)) with VCap cells resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33A-2] Co-culturing transduced CAR-T cells (CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure)) with VCap cells resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33B-1] Co-culturing transduced CAR-T cells (CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure)) with VCap cells resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33B-2] Co-culturing transduced CAR-T cells (CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure)) with VCap cells resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 34A-1] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34A-2] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34B-1] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34B-2]The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 35] The percentage of proliferating cells in co-cultures of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or CAR-T cells transduced from CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) is shown after co-culturing with VCAP or DU145 cells for 5 days. UTD: Untransduced. [Figure 36A-1] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36A-2] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36B-1] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36B-2]The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 37] The figures show the percentage of non-transduced T cells (UTDs) expressing CTV, or CAR-T cells transduced to CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure), after co-culture with VCaP or DU145 (T cells only) for 5 days or stimulation with CD2 / 28 beads. [Figure 38A] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38B] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38C]Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38D] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38E] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38F]Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 39A] This report describes the in vivo efficacy and cytokine profiles of KLK2×CD3 bispecific antibodies in a VCaP xenograft mouse model. Three KLK2×CD3 bispecific antibodies were tested at three dose levels: 5 mg / kg, 1 mg / kg, and 0.2 mg / kg. Tumor growth inhibition was plotted based on tumor volume measurements. [Figure 39B-1] This study describes the in vivo efficacy and cytokine profiles of a KLK2 × CD3 bispecific antibody in a VCaP xenograft mouse model. A 100 μL blood sample was collected from animals via post-orbital hemorrhage 6 hours after the first dose. Plasma was separated from the blood sample by high-speed centrifugation. Luminex assays were performed to quantify IFN- and TNF-α concentrations at various KLK2 bispecific doses. [Figure 39B-2] This study describes the in vivo efficacy and cytokine profiles of a KLK2 × CD3 bispecific antibody in a VCaP xenograft mouse model. A 100 μL blood sample was collected from animals via post-orbital hemorrhage 6 hours after the first dose. Plasma was separated from the blood sample by high-speed centrifugation. Luminex assays were performed to quantify IFN- and TNF-α concentrations at various KLK2 bispecific doses.

[0044] (Detailed description) The methods disclosed can be more readily understood by referring to the following detailed description made in relation to the accompanying drawings, which form part of this disclosure. It should be understood that the methods disclosed are not limited to any particular methods described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate specific embodiments by example and are not intended to limit them to the methods described in the claims.

[0045] All patents, published patent applications, and publications referenced herein are incorporated by reference in the same manner as if they were included herein in their entirety.

[0046] Where a list is presented, please understand that, unless otherwise specified, each individual element of that list and all combinations of that list represent a distinct embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0047] When used herein and in the attached "Claims," ​​the singular forms "a," "an," and "the" include plural references unless specifically indicated otherwise. For example, the reference "a cell" includes combinations of two or more cells, and similar combinations.

[0048] The transitional phrases “comprising,” “consisting essentially of,” and “consisting of” are intended to imply the generally accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any element, process, or component not specified in the claims; and (iii) “consisting essentially of” limits the scope of the claims to specified materials or processes, and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with respect to the phrase “comprising” (or its equivalent) also provide embodiments that are described independently with respect to “consisting essentially of” and “consisting essentially of.”

[0049] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art, which depends to some extent on the method by which the value is measured or determined, i.e., on the limitations of the measurement system. Unless otherwise expressly stated in the examples or elsewhere in the specification in the context of a particular assay, result, or embodiment, "approximately" means that a value is within the greater of one standard deviation or a range of up to 5% in accordance with the practices of the art.

[0050] "Activation," "stimulation," "activated," or "stimulated" refers to the induction of a change in the biological state of a cell that results in the expression of activation markers, cytokine production, or mediation of target cell proliferation or cytotoxicity. Cells can be activated by a primary stimulatory signal. Co-stimulatory signals can amplify the magnitude of the primary signal and suppress cell death after initial stimulation, resulting in a more durable activated state and, consequently, higher cytotoxicity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, induces the proliferation of T cells and / or NK cells, and / or the upregulation or downregulation of key molecules.

[0051] An "alternative scaffold" refers to a single-strand protein framework containing a structured core that associates with a variable domain that has a high tolerance for higher-order structures. Because the variable domain allows for diversity to be introduced without compromising the integrity of the scaffold, the variable domain can be genetically engineered and selected to bind to specific antigens.

[0052] "Antibody-dependent cell-mediated cytotoxicity," or "ADCC," refers to a mechanism by which antibody-coated target cells induce cell death in a manner dependent on their interaction with effector cells possessing lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via the Fc gamma receptor (FcγR) expressed on effector cells.

[0053] "Antibody-dependent cell phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by phagocytic cells such as macrophages or dendritic cells.

[0054] An "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, a part thereof, or a combination thereof) to which an antigen-binding domain or T cell receptor can bind, which can mediate an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens may be expressed by genes from biological samples such as tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or lysates; they may be synthesized from such samples; or they may be purified from such samples.

[0055] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic polypeptides, enzymatically available polypeptides, or genetically engineered polypeptides, and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelid VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDR of an antibody, such as the FR3-CDR3-FR4 moiety, HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to antigens, and multispecific proteins containing antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked to each other via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain, such as a single-chain Fv (scFv) or diabody, when the VH and VL domains are expressed as separate single-chain units. Antigen-binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, for genetic engineering of bispecific and multispecific proteins.

[0056] The term "antibody" has a broad meaning and includes monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; multispecific antibodies such as bispecific, tripspecific, and quadrupspecific antibodies; dimeric, tetrameric, or multimeric antibodies; single-chain antibodies; domain antibodies; and immunoglobulin molecules, including any other modified structures of immunoglobulin molecules containing antigen-binding sites of the required specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and a multimer thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further classified into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chains of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0057] "Bispecificity" refers to a molecule (such as an antibody) that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific molecules may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, for example, cynomolgus monkeys (Macaca cynomolgus, cyno) or chimpanzees (Pan troglodytes), or they may bind to epitopes shared between two or more different antigens.

[0058] "Bispecific anti-hK2 / anti-CD3 antibody," "hk2 / CD3 antibody," "hk2×CD3 antibody," and "anti-hK2 / anti-CD3 protein" refer to antibodies that bind to hk2 and CD3, and include at least one binding domain that specifically binds to hK2 and at least one binding domain that specifically binds to CD3. The domains that specifically bind to hK2 and CD3 are typically V H / V L They are paired. A bispecific anti-hk2×CD3 antibody may be monovalent with respect to binding to either hk2 or CD3.

[0059] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0060] As used herein, a "chimeric antigen receptor (CAR)" is defined as a cell surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are combinations not found together in a single protein in nature. This includes receptors in which the extracellular domain and intracellular signaling domain are not found together in a single receptor protein in nature. CARs are primarily intended for use in lymphocytes such as T cells and natural killer (NK) cells.

[0061] Complement-dependent cell-mediated cytotoxicity (CDC) refers to a mechanism by which the Fc effector domain of a target-bound protein binds to and activates complement component C1q, which then activates the complement cascade, leading to the death of the target cell. Complement activation can also result in the deposition of complement components on the target cell surface, facilitating CDC through the binding of complement receptors (e.g., CR3) to leukocytes.

[0062] The "complementarity-determining region" (CDR) is the antibody region that binds to the antigen. There are three CDRs in the VH (Very High Hormone) (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (Very Low Hormone) (LCDR1, LCDR2, LCDR3). CDR can be defined using various descriptions, including: Kabat (Wu et al. (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton J Bmol Biol 263:800-15, 1996). Correspondence between various descriptions and variable area numbering is described (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). CDRs can be depicted using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs as defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated in the specification.

[0063] "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) complex, consisting of homodimers or heterodimers formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta, and CD3 gamma. Human CD3 epsilon contains the amino acid sequence of SEQ ID NO: 442. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide, or protein fragment unless expressly specified as being from a non-human species. Thus, "CD3" means human CD3 unless specified as being from a non-human species, such as "mouse CD3" or "monkey CD3."

[0064] Throughout this specification, "CD3-specific," "specifically binding to CD3," or "anti-CD3 antibody" refers to an antibody that specifically binds to the CD3-epsilon polypeptide (SEQ ID NO: 442), including antibodies that specifically bind to the CD3-epsilon extracellular domain (ECD) (SEQ ID NO: 443). CD3-ε, together with CD3-γ, -δ, and -ζ, and T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor-CD3 complex. This complex plays a crucial role in antigen binding, recognizing several intracellular signaling pathways. The CD3 complex mediates signaling, leading to T cell activation and proliferation. CD3 is essential for the immune response.

[0065] Sequence ID 442 (Human CD3 epsilon) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS KPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0066] Sequence ID 443 (Human CD3 epsilon extracellular domain) DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0067] "Decrease," "decrease," "attenuate," "reduce," or "mitigate" generally refers to the ability of a test molecule to mediate a lesser response (i.e., a downstream effect) compared to the response mediated by the control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. A decrease may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or a decrease of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, for example, 1.5, 1.6, 1.7, 1.8, etc.).

[0068] "Differentiation" refers to a method of reducing the capacity or proliferation of cells, or of transitioning cells to a more developmentally limited state.

[0069] "Code" or "encoding" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, and the biological properties derived therefrom, which have either a predetermined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a predetermined sequence of amino acids, and which function as a template for the synthesis of other polymers and macromolecules in a biological process. Thus, in a cell or other biological system, if the transcription and translation of mRNA corresponding to a gene produces a protein, then that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to that of the mRNA sequence, and the non-coding strand used as a template for the transcription of the gene or cDNA, can be referred to as encoding a protein or other product of that gene or cDNA.

[0070] "Enhance," "promote," "increase," "expand," or "improve" generally refers to the ability of a test molecule to mediate a greater response (i.e., a downstream effect) compared to the response mediated by the control or vehicle. Exemplary responses include T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or the binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function such as enhanced ADCC, CDC, and / or ADCP. Enhancement may be a statistically significant difference in the measured response between the test molecule and the control (or vehicle), or an increase of approximately 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 times or more, for example, 500, 600, 700, 800, 900, or 1000 times or more (including all integers greater than 1 and decimals in between, for example, 1.5, 1.6, 1.7, 1.8, etc.).

[0071] An "epitope" refers to the portion of an antigen to which an antibody specifically binds. Typically, an epitope consists of a surface population of chemically active (polar, nonpolar, or hydrophobic, etc.) regions, such as amino acids or polysaccharide side chains, and may possess specific three-dimensional structural and charge properties. Epitopes can be composed of continuous and / or discontinuous amino acids that form higher-order structural spatial units. In discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen become very close in three-dimensional space due to protein molecule folding. Antibody "epitopes" differ depending on the methodology used to identify them.

[0072] "Expansion" refers to the result of cell division and cell death.

[0073] "Expressed" and "expression" refer to the well-known transcription and translation that occur intracellularly or in vitro. Expression products, such as proteins, are thus expressed by cells or in vitro and may be intracellular, extracellular, or transmembrane proteins.

[0074] An "expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to guide the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0075] "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)).

[0076] "Fab" or "Fab fragment" refers to an antibody fragment composed of VH, CH1, VL, and CL domains.

[0077] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide crosslinks within a hinge region.

[0078] "Fd" or "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains.

[0079] "Fv" or "Fv fragment" refers to an antibody fragment consisting of a VH domain and a VL domain from a single arm of the antibody. Fv fragments lack the constant region of the Fab(CH1 and CL) region. The VH and VL in the Fv fragment are held together by non-covalent interactions.

[0080] The "Fc" polypeptide in dimer Fc refers to one of the two polypeptides that form the dimer Fc domain. For example, the FC polypeptide in dimer IgG FC contains the constant domain sequences of IgG CH2 and IgG CH3.

[0081] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, as well as a polymer of these (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further divided into hypervariable regions called complementarity determining regions (CDRs) scattered within a framework region (FR). Each VH and VL consists of three CDR and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0082] "Genetic modification" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, resulting in the host cell expressing the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences operably ligated to a polynucleotide encoding a chimeric antigen receptor, such as start, stop, promoter, signaling, secretion, or other sequences used by the cell's genetic mechanisms. The gene or sequence may include non-functional sequences that have no known function. A host cell that receives and expresses the introduced DNA or RNA is "genetically engineered." The DNA or RNA introduced into a host cell may originate from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0083] "Heterogeneous" refers to two or more polynucleotides or polypeptides that are not found in nature in the same relationship to one another.

[0084] "Heterogeneic polynucleotide" refers to a polynucleotide that does not exist in nature and encodes two or more neoantigens as described herein.

[0085] "Heterogeneous polypeptide" refers to a polypeptide that does not exist in nature and contains two or more neoantigen polypeptides as described herein.

[0086] A "host cell" refers to any cell containing heterologous nucleic acids. An example of heterologous nucleic acid is a vector (e.g., an expression vector).

[0087] A “human antibody” refers to an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody includes heavy-chain and light-chain variable regions that “derive” from a human-derived sequence. Such exemplary systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. A “human antibody” typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the human antibody and the system used to obtain the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain, for example, a consensus framework sequence obtained from human framework sequence analysis described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0088] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0089] "In combination" means administering two or more therapeutic agents to a subject together as a mixture, simultaneously as single agents, or sequentially as single agents in any order.

[0090] The "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. This is the functional part of a protein that acts by transmitting information within the cell to regulate cellular activity via signaling pathways defined by generating second messengers, or by acting as an effector in response to such messengers. Intracellular signaling domains generate signals that enhance the immune effector function of CAR-containing cells (e.g., CAR-T cells).

[0091] "Isolation" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system in which molecules are produced, such as recombinant cells, in addition to proteins subjected to at least one purification or isolation step. "Isolated" means molecules that are substantially free from other cellular material and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0092] "Kallikrein-related peptidase 2" or "hK2" refers to a known protein also called kallikrein-2, granular kallikrein 2, or HK2. hK2 is produced as a preproprotein and is cleaved during proteolysis to produce an active protease. All hK2 isoforms and variants are encompassed under "hK2". The amino acid sequences of the various isoforms can be searched using GenBank accession numbers NP_005542.1, NP_001002231.1, and NP_001243009. The full-length human hK2 amino acid sequence in SEQ ID NO: 62 is shown. The sequence includes the signal peptide (residues 1-18) and the propeptide region (residues 19-24).

[0093] Sequence ID 62 MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWV LTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHD LMLLRLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLS NDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKP AVYTKVVHYRKWIKDTIAANP

[0094] "Modifying" refers to either an enhanced or reduced ability of a test molecule to mediate a greater or lesser response (i.e., a downstream effect) compared to a response mediated by a control or vehicle.

[0095] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules (i.e., individual antibodies constituting the population are identical except for possible known modifications such as removal of C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or amidogenesis, methionine oxidation, or asparagine or glutamine amidogenesis). Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0096] "Multispecificity" refers to molecules such as antibodies that specifically bind to two or more different antigens or two or more different epitopes within the same antigen. Multispecific molecules may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, for example, cynomolgus monkeys (Macaca fascicularis, cyno) or chimpanzees (Pan troglodytes), or they may bind to epitopes shared between two or more different antigens.

[0097] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. NK cells are CD16 + CD56 + and / or CD57 + TCR - NK cells refer to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0098] When used in relation to nucleic acids or amino acids, "operably linked" and similar terms refer to the operational linking of nucleic acid sequences or amino acid sequences that are functionally related to each other. For example, operable linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA) and, in some cases, the production of polypeptides (i.e., expression of open reading frames). An operable linked peptide refers to a peptide in which the functional domains of the peptide are positioned at appropriate distances from each other to confer the intended function of each domain.

[0099] The term "paratope" refers to a region or area of ​​an antibody molecule containing residues that are involved in antigen binding and interact with the antigen. A paratope can consist of continuous and / or discontinuous amino acids that form higher-order structural spatial units. The paratope of a given antibody can be defined and characterized in detail at various levels using a variety of experimental and computational methods. Experimental methods include hydrogen / deuterium exchange mass spectrometry (HX-MS). Paratopes are defined differently depending on the mapping method used.

[0100] "Combination of pharmaceuticals" refers to a combination of two or more active ingredients administered together or separately.

[0101] "Pharmaceutical composition" refers to a composition obtained by combining an active ingredient and a pharmaceutically acceptable carrier.

[0102] A "pharmaceutically acceptable carrier" or "excipient" refers to a component in a pharmaceutical composition other than the active ingredient that is non-toxic to the target. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers, or preservatives.

[0103] A "polynucleotide" or "nucleic acid" refers to a synthetic molecule containing nucleotide chains covalently bonded by a sugar-phosphate backbone or other equivalent covalent chemical action. cDNA is a typical example of a polynucleotide. Polynucleotides can be DNA or RNA molecules.

[0104] "Preventing," "preventing," "prevention," or "preventive measures" for a disease or disability means preventing the occurrence of the disability in the subject.

[0105] "Proliferation" refers to an increase in cell division, whether symmetrical or asymmetrical.

[0106] A "promoter" refers to the minimum sequence required to initiate transcription. Promoters may also contain enhancer or repressor elements, respectively, which either enhance or suppress transcription.

[0107] "Protein" and "polypeptide" are used interchangeably herein and refer to molecules comprising one or more polypeptides, each composed of at least two amino acid residues linked by peptide bonds. Proteins may be monomers or protein complexes of two or more identical or different subunits. Small polypeptides consisting of fewer than 50 amino acids may be referred to as "peptides." Proteins may be heterofusion proteins, glycoproteins, or proteins modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation. Proteins may be recombinantly expressed.

[0108] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules prepared, expressed, produced, or isolated by recombinant means.

[0109] "Regulatory element" refers to any cis- or trans-acting genetic element that controls some aspect of nucleic acid sequence expression.

[0110] "Recurrent" refers to the recurrence of a disease or its signs and symptoms after a period of improvement following pre-treatment with a therapeutic agent.

[0111] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before treatment or at the start of treatment, or a refractory disease may become a resistant disease during treatment.

[0112] "Single-chain Fv" or "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein the VL and VH are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in either order. For example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may comprise VL-linker-VH or VH-linker-VL.

[0113] "(scFv)2" or "tandem scFv" or "bis-scFv" fragment is a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), wherein the two VL and two VH regions are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. The two VL regions and two VH regions fused by the peptide linker form a bivalent molecule VL A -linker-VH A -linker-VL B -linker-VH B to form two binding sites capable of simultaneously binding to two different antigens or epitopes.

[0114] "Specifically binding," "specific binding," "specifically bound," or "binding" refers to a protein molecule binding to an antigen or an epitope within that antigen with a higher affinity than its affinity to other antigens. Typically, protein molecules bind to an antigen with an affinity of approximately 1 × 10⁻⁶. -7 M or less, for example, about 5 × 10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or approximately 1 × 10 -12 Equilibrium dissociation constants (K) less than or equal to M D ) binds to the antigen or an epitope within the antigen, and typically, the K D This refers to K for binding to nonspecific antigens (e.g., BSA, casein). D It is at least 100 times smaller than [the specified value]. In the context of the prostatic neoantigen described herein, “specific binding” means that a protein molecule binds to the prostatic neoantigen without the prostatic neoantigen binding to the wild-type protein, which is its variant, in a detectable manner.

[0115] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. The terms "subject" and "patient" may be used interchangeably herein.

[0116] "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells are helper T cells (HTL;CD4 + T cells), CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4 + CD8 + This may be T cells, or any other subset of T cells. It also includes "NKT cells," which refer to a special population of T cells that not only express the semi-variant αβ T cell receptor but also various molecular markers typically associated with NK cells, such as NK1.1. Examples of NKT cells include NK1.1 + and NK1.1 - , and CD4 + CD4 - CD8 + These include NKT cells and CD8 cells. NKT cells are unique in that their TCR recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma-delta T cells (γδT cells)," which refer to a special population that is a small subset of T cells with a different TCR on its surface. Unlike most T cells, whose TCR consists of two glycoprotein chains named α- and β-TCR chains, the TCR in γδT cells consists of a γ chain and a δ chain. γδT cells can play a role in immune surveillance and immune regulation, and are a vital source of IL-17 and robust CD8 cells. + It has been found that these cells induce cytotoxic T cell responses. The term also includes "regulatory T cells" or "Tregs," which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4 cells. + It is a T cell, and also an IL-10 producing CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0117] In this specification, the terms "therapeutic effective dose" or "effective dose" as used interchangeably refer to the amount effective in achieving the desired therapeutic outcome in the required dosage and duration. The therapeutically effective dose may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient's health, reduction in tumor volume, cessation or slowing of tumor growth, and / or absence of metastasis of cancer cells to other parts of the body.

[0118] "Transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0119] "To treat," "to treat," or "to treat" a disease or disability such as cancer means achieving one or more of the following: reducing the severity and / or duration of the disability; inhibiting the exacerbation of symptoms characteristic of the disability being treated; limiting or preventing recurrence of the disability in a person who previously had the disability; or limiting or preventing recurrence of symptoms in a person who previously had the disability.

[0120] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, ex vivo, or tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can be induced by infection with transforming viruses and the incorporation of new genomic nucleic acids, or by the uptake of exogenous nucleic acids, and may occur spontaneously or after exposure to carcinogens, thereby resulting in mutations of endogenous genes. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts such as nude mice, in vitro, in vivo, and ex vivo.

[0121] "Variant," "mutant," or "change" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0122] Throughout this specification, unless otherwise explicitly stated herein, the numbering of amino acid residues in the constant region of an antibody follows the EU index described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0123] Mutations in the Ig constant region are denoted as follows: L351Y_F405A_Y407V refers to L351Y, F405A, and Y407V mutations in one immunoglobulin constant region. L351Y_F405A_Y407V / T394W refers to L351Y, F405A, and Y407V mutations in the first Ig constant region and T394W mutation in the second Ig constant region present in one multimeric protein.

[0124] "VHH" refers to a single-domain antibody or nanobody that is exclusively composed of the antigen-binding domain of the heavy chain. VHH single-domain antibodies lack the CH1 domains of the light and heavy chains in the conventional Fab region.

[0125] composition of a substance hK2-binding antigen-binding domain This disclosure provides an antigen-binding domain that binds to hK2, monospecific and multispecific proteins (particularly bispecific proteins) containing an antigen-binding domain that binds to hK2, chimeric antigen receptors (CARs) containing an antigen-binding domain that binds to hK2, polynucleotides encoding the foregoing, vectors, host cells, and methods for producing and using the foregoing. The hK2-binding antigen-binding domains identified herein have shown improved properties in terms of improved thermal stability. The multispecific proteins disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting increased antitumor effects.

[0126] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 binds to an epitope on hK2 as described in SEQ ID NOs. 111, 112, 113, 114, or 115.

[0127] In certain embodiments, the antigen-binding domain that binds to hK2 binds to the epitope on hK2 described in SEQ ID NOs: 111 and 112.

[0128] In some embodiments, the antigen-binding domain that binds to hK2 binds to the epitope on hK2 described in SEQ ID NOs: 113, 114, and 115.

[0129] The present invention also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein an antibody or its antigen-binding fragment binds within the hK2 residues KVTEF (SEQ ID NO: 111), HYRKW (SEQ ID NO: 112), SHGWAH (SEQ ID NO: 113), RHNLFEPEDTGQRVP (SEQ ID NO: 114), or GWGSIEPEE (SEQ ID NO: 115). In certain embodiments, the present invention also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain binds to hK2 within an epitope having the sequences KVTEF (SEQ ID NO: 111) and HYRKW (SEQ ID NO: 112).

[0130] An H / D exchange assay can be used to determine the residue in hK2 to which the antibody binds. In the H / D exchange assay, recombinantly expressed soluble hK2 is incubated in deuterated water for a predetermined time, either in the presence or absence of the antibody, resulting in deuterium incorporation at an exchangeable hydrogen atom not protected by the antibody. This is followed by protease digestion of the protein and analysis of the peptide fragment using LC-MS. The H / D exchange assay can be performed using known protocols. An exemplary protocol is described in Example 3. In some embodiments, the H / D exchange mixture is quenched by adding a quenching buffer (e.g., 8M urea, 1M TCEP, pH 3.0) and then passed through an immobilized pepsin / FpXIII column equilibrated at room temperature (e.g., 600 μL / min). Next, the digestible fragments are loaded onto a reverse-phase trap column (e.g., 600 μL / min), desalted (e.g., 600 μL for 1 minute), separated (e.g., on a C18 column), and analyzed by mass spectrometry (e.g., using an LTQ® Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with a capillary temperature of 275°C, a resolution of 150,000, and a mass range (m / z) of 300–1,800).

[0131] The present invention also provides an isolated protein comprising an antigen-binding domain, wherein the antigen-binding domain of a reference antibody binds to an epitope on hK2 having a sequence selected from the group consisting of SEQ ID NOs: 111, 112, 113, 114, and 115, and the antigen-binding fragment that binds to hK2 competes with the reference antibody disclosed herein for binding to hK2. In some embodiments, the reference antibody includes: a. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 138; or b. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 163; or c. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 164, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 165; or d. HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or e. HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or f. HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 204, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 205; or g. HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 444.

[0132] In certain such embodiments, the reference antibody includes the heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162, and the light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0133] Competition between the test antibody and the reference antibody of the present invention for binding to soluble hK2 (SEQ ID NOs. 111, 112, 113, 114, or 115) can be assayed in vitro using well-known methods. For example, the binding of the labeled antibody to the membrane-proximal region of hK2 in the presence of an unlabeled reference antibody may be evaluated by ELISA, or competition may be demonstrated using Biacore analysis or flow cytometry. The test antibody competes with the reference antibody for binding to hK2 when it inhibits the binding of the reference antibody to soluble hK2 by 85% or more, for example, 90% or more, or 95% or more.

[0134] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises the following: Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 137, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163; or HC of the VH of SEQ ID NO: 164 DR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205. In certain embodiments, the isolated protein comprises an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), the antigen-binding domains that bind to hK2 include HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 163.

[0135] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the following: HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3: Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively. Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0136] In certain embodiments, the disclosure provides isolated proteins comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively.

[0137] In another embodiment, the disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 194, 195, 172, 173, 174, and 175.

[0138] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises VH of SEQ ID NOs. 137, 162, 164, 166, 168, or 204 and VL of SEQ ID NOs. 138, 163, 165, 167, or 169.

[0139] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the following: VH of sequence number 137 and VL of sequence number 138; VH of sequence number 162 and VL of sequence number 163; VH of sequence number 164 and VL of sequence number 165; VH of sequence number 166 and VL of sequence number 167; VH of sequence number 168 and VL of sequence number 169; or VH of sequence number 204 and VL of sequence number 205.

[0140] In certain embodiments, the disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0141] The disclosure also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162, and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0142] In some embodiments, the antigen-binding domain that binds to hK2 includes, for example, VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 162.

[0143] In some embodiments, the antigen-binding domain that binds to hK2 includes, for example, the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL.

[0144] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 95% identical to the VH of SEQ ID NO: 162 and a VL that is at least 95% identical to the VL of SEQ ID NO: 163.

[0145] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 95% identical to the VH of SEQ ID NO: 162 and a VL that is at least 99% identical to the VL of SEQ ID NO: 163.

[0146] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 99% identical to the VH of SEQ ID NO: 162 and a VL that is at least 99% identical to the VL of SEQ ID NO: 163.

[0147] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 99% identical to the VH of SEQ ID NO: 162 and a VL that is at least 95% identical to the VL of SEQ ID NO: 163.

[0148] The present disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NO: 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409. In certain embodiments, the present disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NO: 404.

[0149] In another embodiment, the present disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NO: 405.

[0150] The present disclosure also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404.

[0151] The present disclosure also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 405.

[0152] The present disclosure also provides an isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK^{2} comprises a heavy-chain variable region (VH) of SEQ ID NO: 75 and a light-chain variable region (VL) of SEQ ID NO: 74. SEQ ID NO: 75 and SEQ ID NO: 74 represent VH and VL amino acid sequences that include variants that exhibit improved thermal stability when compared to the parental antibody hu11B6. The positions that are genetically engineered to confer improved thermal stability were residues P41, I49, M70, and A88 in VH (numbered according to the residues in hu11B6_VH of SEQ ID NO: 5) and S80, L82, A88, and Y91 in VL (numbered according to the residues in hu11B6_VL of SEQ ID NO: 2).

[0153] JPEG0007843226000001.jpg21159

[0154] JPEG0007843226000002.jpg20167

[0155] The present disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises a VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and a VL of SEQ ID NO: 1, 2, 3, 140, or 160, provided that the antigen-binding domain that binds to hK2 does not comprise both a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 2.

[0156] The present disclosure also provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises: VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160; VH of sequence number 139 and VL of sequence number 1; VH of sequence number 139 and VL of sequence number 2; VH of sequence number 139 and VL of sequence number 3; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 139 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 1; VH of sequence number 159 and VL of sequence number 2; VH of sequence number 159 and VL of sequence number 3; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 161 and VL of sequence number 1; VH of sequence number 161 and VL of sequence number 2; VH of sequence number 161 and VL of sequence number 3; VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or VH of sequence number 161 and VL of sequence number 160.

[0157] This disclosure provides an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NOs. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 135, 136, 318, 319, 320, 321, 322, or 323.

[0158] In some embodiments, the antigen-binding domain that binds to hK2 is an scFv.

[0159] In some embodiments, the antigen-binding domain that binds to hK2 is (scFv)2.

[0160] In some embodiments, the antigen-binding domain that binds to hK2 is an Fv.

[0161] In some embodiments, the antigen-binding domain that binds to hK2 is a Fab.

[0162] In some embodiments, the antigen-binding domain that binds to hK2 is F(ab’)2.

[0163] In some embodiments, the antigen-binding domain that binds to hK2 is an Fd.

[0164] In some embodiments, the hK2 antigen-binding domain is a dAb.

[0165] In some embodiments, the hK2 antigen-binding domain is a VHH.

[0166] In certain embodiments, the antigen-binding domain that binds to hK2 is a Fab.

[0167] hK2-binding scFv Either the VH domain or VL domain identified herein that binds to hK2 can be genetically engineered into either the VH-linker-VL or VL-linker-VH orientation of scFv formats. Furthermore, either the VH or VL domain identified herein can be used to construct, for example, the following sc(Fv)2 structures: VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VL-linker-VL, VL-linker-VH-linker-VH-linker-VL, or VL-linker-VL-linker-VH-linker-VH.

[0168] The VH and VL domains identified herein can be incorporated into the scFv format, and the binding and thermal stability of the resulting scFv to hK2 can be evaluated using known methods. Binding can be evaluated using ProteOn XPR36, Biacore3000, or KinExA instruments, ELISA, or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFv or lysed cells containing E. coli supernatant or expressed scFv. Measured affinity of test scFV to hK2 may differ when measured under different conditions (e.g., molar osmotic concentration, pH). Therefore, affinity and other binding parameters (e.g., K) may differ. D , K on , K off The measurement of thermal stability is typically performed using standardized conditions and standardized buffers. Thermal stability can be evaluated by heating a test scFv at a high temperature such as 50°C, 55°C, or 60°C for a period of time such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, and measuring the binding of the test scFv to hK2. An scFv that retains a similar binding to hK2 when compared to an unheated scFv sample is said to be thermally stable.

[0169] In recombinant expression systems, the linker is a peptide linker and may contain any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker needs to be of an appropriate length to link VH and VL in such a way that they form precise higher-order structures relative to each other, in order to maintain desired activities such as binding to hK2.

[0170] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker is approximately 10 to 40 amino acids long. In some embodiments, the linker is approximately 10 to 35 amino acids long. In some embodiments, the linker is approximately 10 to 30 amino acids long. In some embodiments, the linker is approximately 10 to 25 amino acids long. In some embodiments, the linker is approximately 10 to 20 amino acids long. In some embodiments, the linker is approximately 15 to 20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acid lengths. In some embodiments, the linker is 16 amino acid lengths. In some embodiments, the linker is 17 amino acid lengths. In some embodiments, the linker is 18 amino acid lengths. In some embodiments, the linker is 19 amino acid lengths. In some embodiments, the linker is 20 amino acid lengths. In some embodiments, the linker is 21 amino acid lengths. In some embodiments, the linker is 22 amino acid lengths. In some embodiments, the linker is 23 amino acid lengths. In some embodiments, the linker is 24 amino acid lengths. In some embodiments, the linker is 25 amino acid lengths. In some embodiments, the linker is 26 amino acid lengths. In some embodiments, the linker is 27 amino acid lengths. In some embodiments, the linker is 28 amino acid lengths. In some embodiments, the linker is 29 amino acid lengths. In some embodiments, the linker is 30 amino acid lengths.In some embodiments, the linker is 31 amino acid lengths. In some embodiments, the linker is 32 amino acid lengths. In some embodiments, the linker is 33 amino acid lengths. In some embodiments, the linker is 34 amino acid lengths. In some embodiments, the linker is 35 amino acid lengths. In some embodiments, the linker is 36 amino acid lengths. In some embodiments, the linker is 37 amino acid lengths. In some embodiments, the linker is 38 amino acid lengths. In some embodiments, the linker is 39 amino acid lengths. In some embodiments, the linker is 40 amino acid lengths. Exemplary linkers that can be used are glycy-rich linkers, glycy and ser-containing linkers, glycy and ala-containing linkers, ala and ser-containing linkers, and other flexible linkers.

[0171] Other linker sequences may include immunoglobulin hinge regions, CL, or CH1 portions derived from immunoglobulin heavy or light chain isotypes. Alternatively, various non-protein polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may be used as linkers. Exemplary linkers that may be used are shown in Table 1. Additional linkers are described, for example, in International Publication No. 2019 / 060695.

[0172] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL) from the N-terminus to the C-terminus.

[0173] In some embodiments, scFv includes VL, L1, and VH from the N-terminus to the C-terminus (VL-L1-VH).

[0174] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0175] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 76.

[0176] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 77.

[0177] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 78.

[0178] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 79.

[0179] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 80.

[0180] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 81.

[0181] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 82.

[0182] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 83.

[0183] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 84.

[0184] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 85.

[0185] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 86.

[0186] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 87.

[0187] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 88.

[0188] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 89.

[0189] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 90.

[0190] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 91.

[0191] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 92.

[0192] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 93.

[0193] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 94.

[0194] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 95.

[0195] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 96.

[0196] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 97.

[0197] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 98.

[0198] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 99.

[0199] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 100.

[0200] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 101.

[0201] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 102.

[0202] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 103.

[0203] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 104.

[0204] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 105.

[0205] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 106.

[0206] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 107.

[0207] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 108.

[0208] [Table 1]

[0209] In certain embodiments, L1 comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0210] In some embodiments, the scFV includes the following: Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDRs) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 164, and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 165; or HC of the VH of SEQ ID NO: 166 DR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 139, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 140.

[0211] In some embodiments, scFv includes the following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively. Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0212] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 141, 142, 143, 144, 145, and 146.

[0213] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 171, 172, 173, 174, and 175.

[0214] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 176, 177, 178, 179, 180, and 181.

[0215] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 183, 184, 185, 186, and 187.

[0216] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 188, 189, 190, 182, 470, and 209.

[0217] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 147, 148, 143, 144, 145, and 146.

[0218] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 141, 142, 143, 144, 145, and 146.

[0219] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 194, 195, 172, 173, 174, and 175.

[0220] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 196, 197, 178, 179, 180, and 181.

[0221] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 198, 199, 184, 185, 186, and 187.

[0222] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 200, 201, 190, 191, 192, and 193.

[0223] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 216, 217, 218, 182, 470, and 209.

[0224] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 63, 65, 66, 67, 69, and 71, respectively.

[0225] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 63, 65, 66, 68, 70, and 71.

[0226] In some embodiments, the scFV includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 72, 73, 66, 67, 69, and 71, respectively.

[0227] In some embodiments, scFV includes VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138.

[0228] In some embodiments, scFV includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0229] In some embodiments, scFV includes VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165.

[0230] In some embodiments, scFV includes VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167.

[0231] In some embodiments, scFV includes VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169.

[0232] In some embodiments, scFV includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205.

[0233] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160.

[0234] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140.

[0235] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140.

[0236] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140.

[0237] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0238] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0239] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0240] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0241] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0242] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0243] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 159 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 160.

[0244] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 161 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0245] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 139 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0246] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 159 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0247] In some embodiments, scFv includes, for example, a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162, and a VL of SEQ ID NO: 163.

[0248] In some embodiments, scFv includes, for example, VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH and VL of SEQ ID NO: 163.

[0249] In some embodiments, scFv includes a VH that is at least 95% identical to the VH of SEQ ID NO: 162 and a VL that is at least 95% identical to the VL of SEQ ID NO: 163.

[0250] In some embodiments, scFv includes a VH that is at least 99% identical to the VH of sequence number 162 and a VL that is at least 95% identical to the VL of sequence number 163.

[0251] In some embodiments, scFv includes a VH that is at least 99% identical to the VH of sequence number 162 and a VL that is at least 99% identical to the VL of sequence number 163.

[0252] In some embodiments, scFv includes a VH that is at least 95% identical to the VH of sequence number 162 and a VL that is at least 99% identical to the VL of sequence number 163.

[0253] In some embodiments, scFv includes the amino acid sequence of SEQ ID NOs: 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409.

[0254] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NOs. 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409.

[0255] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404.

[0256] In some embodiments, scFv includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 405.

[0257] In some embodiments, scFV includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0258] In some embodiments, scFV includes VH of SEQ ID NO: 4, 5, or 6 and VL of SEQ ID NO: 1, 2, or 3.

[0259] In some embodiments, the scFV comprises the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, provided that the antigen-binding domain that binds to hK2 does not include both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0260] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0261] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0262] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0263] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140.

[0264] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160.

[0265] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0266] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0267] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140.

[0268] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160.

[0269] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0270] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0271] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0272] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140.

[0273] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160.

[0274] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1.

[0275] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2.

[0276] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3.

[0277] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140.

[0278] In some embodiments, scFV includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160.

[0279] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1.

[0280] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2.

[0281] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3.

[0282] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140.

[0283] In some embodiments, scFV includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160.

[0284] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1.

[0285] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2.

[0286] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3.

[0287] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140.

[0288] In some embodiments, scFV includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[0289] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 8.

[0290] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 9.

[0291] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 10.

[0292] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 11.

[0293] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 12.

[0294] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 13.

[0295] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 14.

[0296] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 15.

[0297] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 16.

[0298] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 17.

[0299] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 18.

[0300] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 19.

[0301] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 20.

[0302] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 21.

[0303] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 22.

[0304] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 23.

[0305] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 135.

[0306] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 136.

[0307] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 318.

[0308] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 319.

[0309] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 320.

[0310] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 321.

[0311] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 322.

[0312] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 323.

[0313] hK2-binding and other antigen-binding domains Either the VH domain or VL domain identified herein that binds to hK2 can be genetically engineered into Fab, F(ab')2, Fd, or Fv format, and their binding to hK2 and thermal stability can be evaluated using the assays described herein.

[0314] In some embodiments, Fab includes the following: Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 162, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 163; HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or Alternatively, HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205.

[0315] In certain embodiments, Fab includes HCDR1, HCDR2, and HCDR3 of VH in SEQ ID NO: 162, and LCDR1, LCDR2, and LCDR3 of VL in SEQ ID NO: 163.

[0316] In some embodiments, the Fab includes the following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0317] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 141, 142, 143, 144, 145, and 146.

[0318] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 171, 172, 173, 174, and 175.

[0319] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 176, 177, 178, 179, 180, and 181.

[0320] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 183, 184, 185, 186, and 187.

[0321] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 188, 189, 190, 191, 192, and 193.

[0322] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 206, 207, 208, 182, 470, and 209.

[0323] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 147, 148, 143, 144, 145, and 146.

[0324] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 194, 195, 172, 173, 174, and 175.

[0325] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 196, 197, 178, 179, 180, and 181.

[0326] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 198, 199, 184, 185, 186, and 187.

[0327] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 200, 201, 190, 191, 192, and 193.

[0328] In some embodiments, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 216, 217, 218, 182, 470, and 209.

[0329] In a particular embodiment, the Fab includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 171, 172, 173, 174, and 175, or sequence numbers 194, 195, 172, 173, 174, and 175.

[0330] In some embodiments, Fab includes VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138.

[0331] In some embodiments, Fab includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0332] In some embodiments, Fab includes VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165.

[0333] In some embodiments, Fab includes VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167.

[0334] In some embodiments, Fab includes VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169.

[0335] In some embodiments, Fab includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205.

[0336] In some embodiments, Fab includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0337] In certain embodiments, Fab includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0338] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0339] In some embodiments, scFv includes, for example, a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162, and a VL of SEQ ID NO: 163.

[0340] In some embodiments, scFv includes, for example, VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH and VL of SEQ ID NO: 163.

[0341] In some embodiments, Fab includes a VH that is at least 95% identical to VH of SEQ ID NO: 162 and a VL that is at least 95% identical to VL of SEQ ID NO: 163.

[0342] In some embodiments, Fab includes a VH that is at least 99% identical to VH of SEQ ID NO: 162 and a VL that is at least 95% identical to VL of SEQ ID NO: 163.

[0343] In some embodiments, Fab includes a VH that is at least 99% identical to VH of SEQ ID NO: 162 and a VL that is at least 99% identical to VL of SEQ ID NO: 163.

[0344] In some embodiments, Fab includes a VH that is at least 99% identical to VH of SEQ ID NO: 162 and a VL that is at least 95% identical to VL of SEQ ID NO: 163.

[0345] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0346] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0347] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0348] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0349] In some embodiments, scFv includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0350] In some embodiments, Fab includes the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, provided that Fab does not include both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0351] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0352] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0353] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0354] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140.

[0355] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160.

[0356] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0357] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0358] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140.

[0359] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160.

[0360] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0361] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0362] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0363] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140.

[0364] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160.

[0365] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1.

[0366] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2.

[0367] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3.

[0368] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140.

[0369] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160.

[0370] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1.

[0371] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2.

[0372] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3.

[0373] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140.

[0374] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160.

[0375] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1.

[0376] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2.

[0377] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3.

[0378] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140.

[0379] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[0380] VH and VL of Fab containing an antigen-binding domain that binds to hK2 can be genetically engineered into Fab-Fc HC (VH-CH1-hinge-CH2-CH3) and Fab-Fc LC (VL-CL) formats, respectively. In certain such embodiments, Fab-Fc LC contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354. In certain embodiments, Fab-Fc HC contains an amino acid sequence that is identical to SEQ ID NO: 354.

[0381] In some embodiments, Fab-Fc HC includes a C-terminal lysine residue (e.g., K477). In some embodiments, Fab-Fc HC includes an amino acid sequence having a C-terminal lysine residue and a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 361. In certain embodiments, Fab-Fc HC includes the amino acid sequence of SEQ ID NO: 361.

[0382] In some embodiments, Fab-Fc LC contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221. In certain embodiments, Fab-Fc LC contains an amino acid sequence that is identical to SEQ ID NO: 221.

[0383] As shown in the examples, particularly suitable antigen-binding domains that bind to hK2 for incorporation into a multispecific construct include Fab-Fc HC having the amino acid sequence of SEQ ID NO: 354 and Fab-Fc LC having the amino acid sequence of SEQ ID NO: 221.

[0384] In some embodiments, F(ab')2 includes the following: Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 162, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 163; HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or Alternatively, HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205.

[0385] In some embodiments, F(ab')2 includes the following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0386] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 141, 142, 143, 144, 145, and 146, respectively.

[0387] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively.

[0388] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 176, 177, 178, 179, 180, and 181, respectively.

[0389] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 183, 184, 185, 186, and 187, respectively.

[0390] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 188, 189, 190, 191, 192, and 193, respectively.

[0391] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 206, 207, 208, 182, 470, and 209, respectively.

[0392] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 147, 148, 143, 144, 145, and 146, respectively.

[0393] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 194, 195, 172, 173, 174, and 175, respectively.

[0394] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 196, 197, 178, 179, 180, and 181.

[0395] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 198, 199, 184, 185, 186, and 187, respectively.

[0396] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 200, 201, 190, 191, 192, and 193, respectively.

[0397] In some embodiments, F(ab')2 includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0398] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138.

[0399] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0400] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165.

[0401] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167.

[0402] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169.

[0403] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205.

[0404] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0405] In some embodiments, F(ab')2 includes the VH of SEQ ID NOs: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NOs: 1, 2, 3, 140, or 160, provided that Fab does not include both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0406] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0407] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0408] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0409] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140.

[0410] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160.

[0411] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0412] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0413] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140.

[0414] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160.

[0415] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0416] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0417] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0418] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140.

[0419] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160.

[0420] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1.

[0421] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2.

[0422] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3.

[0423] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140.

[0424] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160.

[0425] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1.

[0426] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2.

[0427] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3.

[0428] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140.

[0429] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160.

[0430] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1.

[0431] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2.

[0432] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3.

[0433] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140.

[0434] In some embodiments, F(ab')2 includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[0435] In some embodiments, Fv includes the following: Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determination regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 162, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 163; HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 165; HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 167; HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 169; or Alternatively, HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and LCDR1, LCDR2, and LCDR3 of VL in sequence number 205.

[0436] In some embodiments, Fv includes the following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0437] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 141, 142, 143, 144, 145, and 146.

[0438] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 170, 171, 172, 173, 174, and 175.

[0439] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 176, 177, 178, 179, 180, and 181.

[0440] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 183, 184, 185, 186, and 187, respectively.

[0441] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 188, 189, 190, 191, 192, and 193.

[0442] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 206, 207, 208, 182, 470, and 209.

[0443] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 147, 148, 143, 144, 145, and 146, respectively.

[0444] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 194, 195, 172, 173, 174, and 175.

[0445] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 196, 197, 178, 179, 180, and 181.

[0446] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 198, 199, 184, 185, 186, and 187, respectively.

[0447] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, with sequence numbers 200, 201, 190, 191, 192, and 193.

[0448] In some embodiments, Fv includes HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0449] In some embodiments, Fv includes VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138.

[0450] In some embodiments, Fv includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0451] In some embodiments, Fv includes VH of sequence number 164 and VL of sequence number 165.

[0452] In some embodiments, Fv includes VH of sequence number 166 and VL of sequence number 167.

[0453] In some embodiments, Fv includes VH of sequence number 168 and VL of sequence number 169.

[0454] In some embodiments, Fv includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205.

[0455] In some embodiments, Fv includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0456] In some embodiments, Fv includes VH of sequence number 4, 5, or 6 and VL of sequence number 1, 2, or 3.

[0457] In some embodiments, Fv includes the VH of sequence number 4, 5, 6, 139, 159, or 161 and the VL of sequence number 1, 2, 3, 140, or 160, provided that Fab does not include both the VH of sequence number 5 and the VL of sequence number 2.

[0458] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0459] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0460] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0461] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140.

[0462] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160.

[0463] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0464] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0465] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140.

[0466] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160.

[0467] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0468] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0469] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0470] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140.

[0471] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160.

[0472] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1.

[0473] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2.

[0474] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3.

[0475] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140.

[0476] In some embodiments, Fv includes VH of sequence number 139 and VL of sequence number 160.

[0477] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1.

[0478] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2.

[0479] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3.

[0480] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140.

[0481] In some embodiments, Fv includes VH of sequence number 159 and VL of sequence number 160.

[0482] In some embodiments, Fv includes VH of sequence number 161 and VL of sequence number 1.

[0483] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2.

[0484] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3.

[0485] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140.

[0486] In some embodiments, Fv includes VH of sequence number 161 and VL of sequence number 160.

[0487] In some embodiments, Fd includes VH of sequence number 75.

[0488] In some embodiments, Fd includes VH of sequence number 4.

[0489] In some embodiments, Fd includes VH of sequence number 5.

[0490] In some embodiments, Fd includes VH of sequence number 6.

[0491] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 210 and the LC of SEQ ID NO: 221.

[0492] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 211 and the LC of SEQ ID NO: 222.

[0493] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 212 and the LC of SEQ ID NO: 223.

[0494] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 213 and the LC of SEQ ID NO: 224.

[0495] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 219 and the LC of SEQ ID NO: 220.

[0496] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment includes an HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354 and an LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221.

[0497] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment includes HC and LC of SEQ ID NO: 221, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354.

[0498] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment includes an HC of SEQ ID NO: 354 and an LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221.

[0499] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises an HC which is at least 95% identical to SEQ ID NO: 354 and an LC which is at least 95% identical to SEQ ID NO: 221.

[0500] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises an HC which is at least 99% identical to SEQ ID NO: 354 and an LC which is at least 95% identical to SEQ ID NO: 221.

[0501] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises an HC which is at least 99% identical to SEQ ID NO: 354 and an LC which is at least 99% identical to SEQ ID NO: 221.

[0502] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises an HC which is at least 95% identical to SEQ ID NO: 354 and an LC which is at least 99% identical to SEQ ID NO: 221.

[0503] In certain embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment comprises the HC of SEQ ID NO: 354 and the LC of SEQ ID NO: 221.

[0504] Homologous antigen-binding domains and antigen-binding domains with conserved substitutions Variants of the antigen-binding domain that binds to hK2 are within the scope of this disclosure. For example, a variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions in the antigen-binding domain that binds to hK2, insofar as it retains or has improved functional properties compared to the parent antigen-binding domain. In some embodiments, sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the antigen-binding domain that binds to hK2 of this disclosure. In some embodiments, the diversity resides in the framework region. In some embodiments, variants are generated by conservative substitution.

[0505] For example, the antigen-binding domain that binds to hK2 may include substitutions at residue positions D16, A23, P41, G45, I49, M70, A88, and V89 in VH (residue numbering according to hu11B6_VH in SEQ ID NO: 5) and at residue positions D1, D9, S10, A12, V13, L15, I21, N22, K24, K49, R58, V62, D64, S80, L82, Q83, A84, V87, A88, Y91, Q104, and L108 in VL (residue numbering according to hu11B6_VL in SEQ ID NO: 2). Conservative substitutions can be made at any designated position, and the resulting variant of the antigen-binding domain that binds to hK2 is tested for desired properties using the assay described herein.

[0506] In some embodiments, the isolated protein containing the antigen-binding domain that binds to hK2 includes VH and VL which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH and VL of the antigen-binding domain that binds to hK2 disclosed herein.

[0507] Furthermore, an antigen-binding domain that binds to hK2 is also provided, which includes VH and VL, which are at least 80% identical to the following. VH of sequence number 137 and VL of sequence number 138; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 161 and VL of sequence number 140; VH of sequence number 162 and VL of sequence number 163; VH of sequence number 164 and VL of sequence number 165; VH of sequence number 166 and VL of sequence number 167; VH of sequence number 168 and VL of sequence number 169; or VH of sequence number 204 and VL of sequence number 205.

[0508] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0509] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0510] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0511] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0512] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0513] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166, and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 444.

[0514] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0515] In some embodiments, the antigen-binding domain that binds to hK2 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0516] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 85% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0517] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 90% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0518] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 91% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0519] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 92% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0520] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 93% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0521] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 94% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0522] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 95% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0523] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 96% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0524] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 97% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0525] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 98% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0526] In some embodiments, the antigen-binding domain that binds to hK2 includes VH, which is at least 99% identical to VH of SEQ ID NO: 162, and VL of SEQ ID NO: 163.

[0527] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 85% identical to VH.

[0528] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 90% identical to VH and VL of SEQ ID NO: 163.

[0529] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 91% identical to VH and VL of SEQ ID NO: 163.

[0530] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 92% identical to VH and VL of SEQ ID NO: 163.

[0531] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 93% identical to VH.

[0532] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 94% identical to VH and VL of SEQ ID NO: 163.

[0533] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 95% identical to VH and VL of SEQ ID NO: 163.

[0534] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 96% identical to VH and VL of SEQ ID NO: 163.

[0535] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 97% identical to VH.

[0536] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 98% identical to VH and VL of SEQ ID NO: 163.

[0537] In some embodiments, the antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which is at least 99% identical to VH and VL of SEQ ID NO: 163.

[0538] The identity percentage between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100).

[0539] The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci 4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid or nucleic acid sequences can also be determined using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453(1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0540] In some embodiments, variants of the antigen-binding domain that bind to hK2 include one or two conservative substitutions in any of the CDR regions while retaining the desired functional properties of the parent antigen-binding fragment that binds to hK2.

[0541] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of antibodies, including amino acid modifications. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative amino acid substitution is a substitution in which one amino acid is replaced by an amino acid residue with a similar side chain. The families of amino acid residues with similar side chains are clearly defined and include amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), non-charged side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), β-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67, Sasaki et al., (1988) Adv Biophys 35:1-24), any native residue within the polypeptide can be substituted with alanine. Amino acid substitutions in the antibodies of the present invention can be performed by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a library of variants can be generated, for example, using random codons (NNK) or non-random codons (e.g., DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)). The resulting variants can be characterized using the assays described herein.

[0542] Method for producing antigen-binding fragments that bind to hK2 The antigen-binding domains that bind to hK2 provided in this disclosure can be produced using a variety of techniques. For example, the Kohler and Milstein hybridoma method can be used to identify VH / VL pairs that bind to hK2. In the hybridoma method, mice or other host animals, such as hamsters, rats, or chickens, are immunized with human and / or cynomolgus monkey hK2, and then hybridoma cells are formed by fusing spleen cells of animals immunized with myeloma cells using standard methods. Colonies arising from a single immortalized hybridoma cell may be screened for the production of antibodies containing antigen-binding domains that bind to hK2 having desired properties such as binding specificity, cross-reactivity or lack thereof, affinity for the antigen, and any desired functionality.

[0543] The antigen-binding domain that binds to hK2 produced by immunizing non-human animals may be humanized. Exemplary humanization techniques, including the selection of human acceptor frameworks, include CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), resurfacing of specificity-determining residues (U.S. Patent Application Publication No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,356), or hyperhumanization (U.S. Patent No. 7,709,226). These methods involve transplanting a CDR or a subset of CDR residues from a parent antibody into a human framework that can be selected based on overall homology to the parent framework, based on similarity in CDR length, identity of canonical structure, or a combination thereof.

[0544] The humanized antigen-binding domain may be further optimized to improve its selectivity or affinity for a desired antigen by incorporating altered framework-supporting residues (reverse mutations) to maintain binding affinity, or by introducing diversity into either the CDR, for example, to improve the affinity of the antigen-binding domain, using techniques such as those described in International Publication Nos. 1090 / 007861 and 1992 / 22653.

[0545] Antigen-binding fragments that bind to hK2 can be produced using transgenic animals such as mice, rats, or chickens that possess a human immunoglobulin (Ig) locus in their own genome, as described, for example, in U.S. Patent No. 6,150,584, International Publication No. 1999 / 45962, 2002 / 066630, 2002 / 43478, 2002 / 043478, and 1990 / 04036. The endogenous immunoglobulin locus of such animals may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal genome using homologous or non-homologous recombination, using a transchromosome, or using a minigene. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_omtinc_net), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) may be working to provide human antibodies targeting selected antigens using the above technologies. In some embodiments, Ablexis mice and OmniRat rats were immunized with soluble full-length KLK2 protein (human kallikrein-2 6-His protein) (SEQ ID NO: 454).

[0546] Antigen-binding domains that bind to hK2 can be selected from phage display libraries in which phages are genetically engineered to express human immunoglobulin or a portion thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions. Antigen-binding domains that bind to hK2 can be isolated from phage display libraries that express antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat proteins, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 09 / 085462. Libraries may be screened for phage binding to human and / or cynomolgus monkey hK2, positive clones obtained may be further characterized, Fab may be isolated from clone lysates and converted to scFV or other components of the antigen-binding fragment.

[0547] The preparation of immunogenic antigens and the expression and generation of the antigen-binding domains of this disclosure can be carried out using any preferred technique, such as recombinant protein generation. Immunogenic antigens may be administered to animals in the form of purified proteins or protein mixtures containing whole cells or cell or tissue extracts, or the antigens may be newly formed (de novo) in the body of the animal from nucleic acids encoding the antigen or a portion thereof.

[0548] Conjugation to the half-life extension portion The antigen-binding domain that binds to hK2 in this disclosure may be conjugated to a half-life extension region. Exemplary half-life extension regions include albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and its fragments and analogs, immunoglobulin (Ig) or its fragments, such as the Fc region. The amino acid sequences of the aforementioned half-life extension regions are known. Ig or its fragments include all isotypes, namely IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0549] Additional half-life extension moieties that can be conjugated to the antigen-binding domain of hK2 of this disclosure include polyethylene glycol (PEG) molecules, e.g., PEG5000 or PEG20000, fatty acids and fatty acid esters of different chain lengths, e.g., laurate, myristic acid, stearate, arachidic acid, behenate, oleate, arachidonic acid, octanodioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, and carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides), for desired properties. These moieties may be directly fused to the antigen-binding domain of hK2 of this disclosure and may be prepared by standard cloning and expression techniques. Alternatively, they may be conjugated to the recombinantly generated antigen-binding domain of hK2 of this disclosure using well-known chemical coupling methods.

[0550] For example, the pegyl moiety can be conjugated to the antigen-binding domain of the present disclosure by incorporating a cysteine ​​residue into the C-terminus of the antigen-binding domain that binds to hK2 of the present disclosure, or by genetically manipulating the cysteine ​​to a residue position facing away from the hK2-binding site, and then attaching a pegyl group to the cysteine ​​using a well-known method.

[0551] In some embodiments, the antigen-binding fragment that binds to hK2 is conjugated to the half-life extension portion.

[0552] In some embodiments, the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, the Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol. In some embodiments, the half-life extension portion is the Ig constant region.

[0553] In some embodiments, the half-life extension portion is Ig.

[0554] In some embodiments, the half-life extension portion is a fragment of Ig.

[0555] In some embodiments, the half-life extension portion is the Ig steady-state region.

[0556] In some embodiments, the half-life extension portion is a fragment of the Ig steady-state region.

[0557] In some embodiments, the half-life extension portion is the Fc region.

[0558] In some embodiments, the half-life extension portion is albumin.

[0559] In some embodiments, the half-life extension portion is the albumin-binding domain.

[0560] In some embodiments, the half-life extension portion is transferrin.

[0561] In some embodiments, the half-life extension portion is polyethylene glycol.

[0562] The antigen-binding domain that binds to hK2, which is conjugated to the half-life extension portion, can be evaluated for its pharmacokinetic properties using known in vivo models.

[0563] Conjugation to the constant region of immunoglobulin (Ig) or fragments of the constant region of Ig The antigen-binding domains of hK2 in this disclosure can be conjugated to an Ig constant region or fragment of an Ig constant region to confer antibody-like properties, including Fc effector function (C1q binding), complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or downregulation of cell surface receptors (e.g., B cell receptors; BCRs). The Ig constant region or fragment of an Ig constant region can also function as half-life extension portions, as discussed herein. The antigen-binding domains of hK2 in this disclosure can be genetically engineered using standard methods to form conventional full-length antibodies. Full-length antibodies containing the antigen-binding domains that bind to hK2 may be further genetically engineered as described herein.

[0564] The constant region of the immunoglobulin heavy chain consists of subdomains CH1, hinge, CH2, and CH3. Following the EU index, the CH1 domain in the heavy chain comprises residues A118–V215, the CH2 domain residues A231–K340, and the CH3 domain residues G341–K447. In some examples, G341 is referred to as the CH2 domain residue. The hinge is generally defined as containing E216 and terminating at P230 in human IgG1. The Ig Fc region includes at least the CH2 and CH3 domains of the Ig constant region and therefore includes at least the region from approximately A231 to K447 of the Ig heavy chain constant region.

[0565] The present invention also provides an antigen-binding domain that binds to an immunoglobulin (Ig) constant region or a fragment of the Ig constant region conjugated to hK2.

[0566] In some embodiments, the Ig steady-state region is the heavy chain steady-state region.

[0567] In some embodiments, the Ig steady-state region is the light chain steady-state region.

[0568] In some embodiments, the Ig steady-state region fragment includes an Fc region.

[0569] In some embodiments, the Ig constant region fragment includes a CH2 domain.

[0570] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0571] In some embodiments, the Ig constant region fragment includes a CH2 domain and a CH3 domain.

[0572] In some embodiments, the Ig constant region fragment includes at least a portion of the hinge, a CH2 domain, and a CH3 domain. The portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0573] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0574] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the N-terminus of the Ig constant region or a fragment of the Ig constant region.

[0575] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the C-terminus of the Ig constant region or a fragment of the Ig constant region.

[0576] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the Ig constant region or a fragment of the Ig constant region via a second linker (L2).

[0577] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

[0578] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 7.

[0579] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 76.

[0580] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 77.

[0581] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 78.

[0582] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 79.

[0583] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 80.

[0584] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 81.

[0585] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 82.

[0586] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 83.

[0587] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 84.

[0588] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 85.

[0589] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 86.

[0590] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 87.

[0591] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 88.

[0592] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 89.

[0593] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 90.

[0594] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 91.

[0595] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 92.

[0596] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 93.

[0597] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 94.

[0598] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 95.

[0599] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 96.

[0600] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 97.

[0601] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 98.

[0602] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 99.

[0603] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 100.

[0604] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 101.

[0605] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 102.

[0606] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 103.

[0607] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 104.

[0608] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 105.

[0609] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 106.

[0610] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 107.

[0611] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 108.

[0612] The antigen-binding domain conjugated to the Ig constant region or a fragment of the Ig constant region of the present disclosure to bind to hK2 can be evaluated for its functionality using several known assays. Binding to hK2 can be evaluated using the methods described herein. The altered properties conferred by the Ig constant region or a fragment of the Ig constant region, such as the Fc region, can be assayed in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or using cell-based assays that measure ADCC, CDC, or ADCP, for example.

[0613] ADCC can be evaluated using an in vitro assay that uses hK2-expressing cells as target cells and NK cells as effector cells. Cell lysis can be detected by the release of a label (e.g., radioactive substrate, fluorescent dye, or native intracellular protein) from the lysed cells. In an exemplary assay, target cells are used in a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and a test antibody. Cell lysis was measured by incubating the sample for 2 hours and measuring the BATDA released into the supernatant. Data were normalized to the maximum cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich), and the minimum control was determined by the spontaneous release of BATDA from target cells in the absence of any antibody.

[0614] ADCP can be evaluated by using monocyte-derived macrophages as effector cells and any hK2-expressing cells genetically engineered to express GFP or other labeled molecules as target cells. In an exemplary assay, the effector:target cell ratio may be, for example, 4:1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the present invention. After incubation, the cells can be detached using actase. Macrophages can be identified by fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the phagocytic percentage can be measured using standard methods for CD11 + and CD14 + This can be determined based on the percentage of GFP fluorescence in macrophages.

[0615] For example, the CDC of cells is 1 × 10⁶ when Doudi cells are treated with RPMI-B (RPMI supplemented with 1% BSA). 5The reaction can be measured by plating cells / well (50 μL / well), adding 50 μL of test protein to the well at a final concentration of 0–100 μg / mL, incubating the reaction at room temperature for 15 minutes, adding 11 μL of pooled human serum to the well, and incubating the reaction at 37°C for 45 minutes. The percentage of lysed cells (%) can be detected as the percentage of propidium iodide-stained cells in the FACS assay using a standard method.

[0616] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the IgG1 heavy chain constant region or a fragment of the IgG1 heavy chain constant region. In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the IgG1 heavy chain constant region or a fragment of the IgG1 heavy chain constant region (e.g., including hinge-CH2-CH3) that contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 110. In some embodiments, the IgG1 heavy chain constant region contains the Fc silencing mutation (L234A_L235A_D265S) and the T350V_T366L_K392L_T394W mutation designed to promote selective heterodimerization. In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to the IgG1 heavy chain constant region or a fragment of the IgG1 heavy chain constant region having the amino acid sequence of SEQ ID NO: 110.

[0617] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 heavy chain constant region (e.g., CH1-hinge-CH2-CH3) containing an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 378. In certain embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 heavy chain constant region or a fragment of the IgG1 heavy chain constant region containing the amino acid sequence of SEQ ID NO: 378.

[0618] In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 light chain constant region containing an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 309. In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 light chain constant region that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 447. In some embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 light chain constant region containing an amino acid sequence that is identical to SEQ ID NO: 309 or 447. In certain embodiments, the antigen-binding domain that binds to hK2 is conjugated to an IgG1 light chain constant region containing an amino acid sequence that is identical to SEQ ID NO: 309.

[0619] In certain embodiments, the isolated protein disclosed herein includes an antigen-binding domain that binds to hK2 conjugated to an IgG1 heavy chain constant region containing the amino acid sequence of SEQ ID NO: 378, and an antigen-binding domain that binds to hK2 conjugated to an IgG1 light chain constant region containing the same amino acid sequence as SEQ ID NO: 309.

[0620] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having an amino acid sequence identical to that of SEQ ID NO: 378.

[0621] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having an amino acid sequence identical to that of SEQ ID NO: 309.

[0622] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378, and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309.

[0623] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), and a second Ig constant region or a fragment of a second Ig constant region having an amino acid sequence identical to that of SEQ ID NO: 109.

[0624] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378, and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309, the isolated protein further comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0625] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, and a first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO. 378.

[0626] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having the same amino acid sequence as SEQ ID NO. 309.

[0627] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NOs. 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NOs. 309.

[0628] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), including HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261, and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0629] In certain embodiments, the isolated protein disclosed herein comprises: a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively; (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO. 378; and (ii) a first Ig light chain comprising the same amino acid sequence as SEQ ID NO. 309. The isolated protein comprises a constant region or a fragment of a first Ig light chain constant region, the isolated protein further comprising a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), the second antigen-binding domain that binds to a lymphocyte antigen, comprising HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261, and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0630] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and a first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378.

[0631] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309.

[0632] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO: 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NO: 309.

[0633] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), comprising the amino acid sequence of SEQ ID NO: 331, and a second Ig constant region or fragment of a second Ig constant region comprising the same amino acid sequence as SEQ ID NO: 109.

[0634] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO: 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NO: 309, wherein the isolated protein further comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), comprising the same amino acid sequence as SEQ ID NO: 331; and a second Ig constant region or fragment of a second Ig constant region comprising the same amino acid sequence as SEQ ID NO: 109.

[0635] Proteins containing an antigen-binding domain that binds to hK2 (as disclosed herein) The antigen-binding domains that bind to hK2 in this disclosure can be genetically engineered into monospecific or multispecific proteins of various designs using standard methods.

[0636] This disclosure also provides a monospecific protein comprising an isolated antigen-binding domain that binds to hK2 of this disclosure.

[0637] In some embodiments, the single-specific protein is an antibody.

[0638] This disclosure also provides a multispecific protein comprising an antigen-binding domain that binds to hK2 of this disclosure.

[0639] In some embodiments, the multispecific protein is bispecific.

[0640] In some embodiments, the multispecific protein is triple specific.

[0641] In some embodiments, the multispecific protein is quadruplespecific.

[0642] In some embodiments, the multispecific protein is monovalent with respect to binding to hK2.

[0643] In some embodiments, the multispecific protein is divalent with respect to binding to hK2.

[0644] This disclosure also provides an isolated, multispecific protein comprising a first antigen-binding domain that binds to hK2 and a second antigen-binding domain that binds to a lymphocyte antigen (such as CD3).

[0645] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0646] In some embodiments, the T cell antigen is CD8 + It is a T cell antigen.

[0647] In some embodiments, the lymphocyte antigen is the NK cell antigen.

[0648] In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0649] In some embodiments, the lymphocyte antigen is CD3ε.

[0650] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0651] In some embodiments, the anti-hK2 / anti-CD3 protein exhibits bispecificity.

[0652] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0653] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen comprises Fab.

[0654] In certain embodiments, the first antigen-binding domain that binds to hK2 includes Fab.

[0655] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes Fab.

[0656] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes F(ab')2.

[0657] In some embodiments, the first antigen-binding domain that binds to hK2 includes F(ab')2.

[0658] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes F(ab')2.

[0659] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen comprises VHH.

[0660] In some embodiments, the first antigen-binding domain that binds to hK2 includes VHH.

[0661] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes VHH.

[0662] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes Fv.

[0663] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fv.

[0664] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes Fv.

[0665] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes Fd.

[0666] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fd.

[0667] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes Fd.

[0668] In some embodiments, the first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes scFV.

[0669] In certain embodiments, the multispecific protein is bispecific, comprising a first antigen-binding domain that binds to hK2, containing Fab, and a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), containing scFv.

[0670] In some embodiments, the first antigen-binding domain to bind includes an scFV.

[0671] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes an scFV.

[0672] In some embodiments, the first antigen-binding domain that binds to hK2 comprises scFV, and the second antigen-binding domain that binds to lymphocyte antigens comprises Fab.

[0673] In certain embodiments, the first antigen-binding domain that binds to hK2 comprises Fab, and the second antigen-binding domain that binds to lymphocyte antigens comprises scFV.

[0674] In some embodiments, the first antigen-binding domain that binds to hK2 comprises scFV, and the second antigen-binding domain that binds to lymphocyte antigens comprises Fab'.

[0675] In some embodiments, the first antigen-binding domain that binds to hK2 comprises Fab', and the second antigen-binding domain that binds to lymphocyte antigens comprises scFV.

[0676] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV, and the second antigen-binding domain that binds to lymphocyte antigens includes Fv.

[0677] In some embodiments, the first antigen-binding domain that binds to hK2 includes dAb, and the second antigen-binding domain that binds to lymphocyte antigens includes scFV.

[0678] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV, and the second antigen-binding domain that binds to lymphocyte antigens includes dAb.

[0679] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fd, and the second antigen-binding domain that binds to lymphocyte antigens includes scFV.

[0680] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV, and the second antigen-binding domain that binds to lymphocyte antigens includes VHH.

[0681] In some embodiments, the first antigen-binding domain that binds to hK2 comprises VHH, and the second antigen-binding domain that binds to lymphocyte antigens comprises scFV.

[0682] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fv, and the second antigen-binding domain that binds to lymphocyte antigens includes scFV.

[0683] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV, and the second antigen-binding domain that binds to lymphocyte antigens includes Fd.

[0684] In some embodiments, scFv includes VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

[0685] In some embodiments, L1 contains approximately 5 to 50 amino acids.

[0686] In some embodiments, L1 contains approximately 5 to 40 amino acids.

[0687] In some embodiments, L1 contains approximately 10 to 30 amino acids.

[0688] In some embodiments, L1 contains approximately 10 to 20 amino acids.

[0689] In some embodiments, L1 includes the amino acid sequence of SEQ ID NOs: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

[0690] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0691] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 76.

[0692] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 77.

[0693] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 78.

[0694] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 79.

[0695] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 80.

[0696] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 81.

[0697] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 82.

[0698] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 83.

[0699] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 84.

[0700] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 85.

[0701] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 86.

[0702] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 87.

[0703] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 88.

[0704] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 89.

[0705] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 90.

[0706] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 91.

[0707] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 92.

[0708] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 93.

[0709] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 94.

[0710] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 95.

[0711] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 96.

[0712] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 97.

[0713] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 98.

[0714] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 99.

[0715] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 100.

[0716] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 101.

[0717] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 102.

[0718] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 103.

[0719] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 104.

[0720] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 105.

[0721] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 106.

[0722] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 107.

[0723] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 108.

[0724] In certain embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0725] In some embodiments, the first antigen-binding domain that binds to hK2 is HCDR1 of sequence numbers 63, 72, 141, 147, 170, 176, 188, 194, 196, 198, 200, 206, or 216, HCDR2 of sequence numbers 64, 65, 73, 142, 148, 171, 177, 188, 189, 195, 197, 199, 201, 207, or 217, sequence number Includes HCDR3 of sequence numbers 66, 143, 172, 178, 184, 190, 208, or 218; LCDR1 of sequence numbers 67, 68, 144, 173, 179, 182, 185, or 191; LCDR2 of sequence numbers 69, 70, 145, 174, 180, 186, 192, or 470; and LCDR3 of sequence numbers 71, 146, 175, 181, 187, 193, or 209.

[0726] In some embodiments, the first antigen-binding domain that binds to hK2 includes the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. Sequence IDs 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 63, 64, 66, 67, 69, and 71, respectively; Sequence IDs 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence IDs 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 72, 73, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0727] In certain embodiments, the first antigen-binding domain that binds to hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively.

[0728] In certain embodiments, the first antigen-binding domain that binds to hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Sequence IDs 170, 171, 172, 173, 174, and 175, respectively, and optionally, a second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0729] In certain embodiments, the first antigen-binding domain that binds to hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Sequence IDs 194, 195, 172, 173, 174, and 175, respectively, and optionally, a second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0730] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138.

[0731] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0732] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165.

[0733] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167.

[0734] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169.

[0735] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205.

[0736] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0737] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0738] In some embodiments, the first antigen-binding domain that binds to hK2 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0739] In some embodiments, the first antigen-binding domain that binds to hK2 includes, for example, VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 162.

[0740] In some embodiments, the first antigen-binding domain that binds to hK2 includes, for example, the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the VL is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162.

[0741] In some embodiments, the first antigen-binding domain that binds to hK2 comprises a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163, and optionally a second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0742] In some embodiments, the first antigen-binding domain that binds to hK2 comprises VH and VL of SEQ ID NO: 163, which are at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 162, and optionally, the second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0743] In some embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical, and optionally, the second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0744] In certain embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0745] In certain embodiments, the first antigen-binding domain that binds to hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and optionally, the second antigen-binding domain that binds to a lymphocyte antigen such as CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example, CD3.

[0746] In some embodiments, the first antigen-binding domain that binds to hK2 includes the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, provided that Fab does not include both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0747] In some embodiments, the first antigen-binding domain that binds to hK2 includes the VH of SEQ ID NO: 4, 5, or 6 and the VL of SEQ ID NO: 1, 2, or 3, provided that the first antigen-binding domain that binds to hK2 does not include both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0748] In some embodiments, the first antigen-binding domain that binds to hK2 includes: VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160; VH of sequence number 139 and VL of sequence number 1; VH of sequence number 139 and VL of sequence number 2; VH of sequence number 139 and VL of sequence number 3; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 139 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 1; VH of sequence number 159 and VL of sequence number 2; VH of sequence number 159 and VL of sequence number 3; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 161 and VL of sequence number 1; VH of sequence number 161 and VL of sequence number 2; VH of sequence number 161 and VL of sequence number 3; VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or VH of sequence number 161 and VL of sequence number 160.

[0749] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 133, 134, 135, 136, 308, 316, 318, 319, 320, 321, 322, 323, 324, 325, 404, 405, 406, 407, 408, or 409.

[0750] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 8.

[0751] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 9.

[0752] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 10.

[0753] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 11.

[0754] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 12.

[0755] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 13.

[0756] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 14.

[0757] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 15.

[0758] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 16.

[0759] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 17.

[0760] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 18.

[0761] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 19.

[0762] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 20.

[0763] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 21.

[0764] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 22.

[0765] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 23.

[0766] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 133.

[0767] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 134.

[0768] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 135.

[0769] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 136.

[0770] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 308.

[0771] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 316.

[0772] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 318.

[0773] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 319.

[0774] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 320.

[0775] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 321.

[0776] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 322.

[0777] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 323.

[0778] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 324.

[0779] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 325.

[0780] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 404.

[0781] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 405.

[0782] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 406.

[0783] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 407.

[0784] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 408.

[0785] In some embodiments, the first antigen-binding domain that binds to hK2 includes the amino acid sequence of SEQ ID NO: 409.

[0786] In some embodiments, the first antigen-binding domain that binds to hK2 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404.

[0787] In some embodiments, the first antigen-binding domain that binds to hK2 includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 405.

[0788] In certain embodiments, the first antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NO: 404 or 405. The disclosure also provides a second antigen-binding domain that binds to a lymphocyte antigen, comprising the heavy chain variable region (VH) of SEQ ID NO: 248 and the light chain variable region (VL) of SEQ ID NO: 157. SEQ ID NOs: 248 and 157 represent the VH and VL amino acid sequences of the parent antibody CD3B815 antibody.

[0789] VL consensus sequence (sequence number 157) DIQX1TQSPX2X3LSX4SX5GX6RVX7X8X9CRARQSIGTAIHWYQQKX 10 X 11 X 12 X 13 PX 14 LLIX 15 YASESISGX 16 PSRFSGSGSGTDFTLTIX 17 SX 18 QX 19 EDX 20 AX 21 YYCQQSX 22 SWPYTFGX 23 GTKLEIK

[0790] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes the following: HCDR1 of sequence number 255, HCDR2 of sequence number 256, HCDR3 of sequence number 257, LCDR1 of sequence number 258, LCDR2 of sequence number 259, and LCDR3 of sequence number 260; or HCDR1 (sequence number 255), HCDR2 (sequence number 256), HCDR3 (sequence number 257), LCDR1 (sequence number 258), LCDR2 (sequence number 259), and LCDR3 (sequence number 261).

[0791] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes the following: VH of sequence number 248 and VL of sequence number 249; or VH of sequence number 248 and VL of sequence number 250; or VH of sequence number 248 and VL of sequence number 251; or VH of sequence number 248 and VL of sequence number 252; or VH of sequence number 248 and VL of sequence number 253; or VH of sequence number 248 and VL of sequence number 254.

[0792] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 251.

[0793] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes, for example, VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251, which are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 248.

[0794] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes, for example, the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251, and the VL being at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248.

[0795] In certain embodiments, the second antigen-binding domain that binds to lymphocyte antigens includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0796] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen includes the following: HCDR1 of sequence number 116, HCDR2 of sequence number 117, HCDR3 of sequence number 118, LCDR1 of sequence number 119, LCDR2 of sequence number 120, and LCDR3 of sequence number 121; or VH of sequence number 122 and VL of sequence number 123.

[0797] In certain embodiments, the second antigen-binding domain that binds to lymphocyte antigens includes HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[0798] In certain embodiments, the first antigen-binding domains that bind to hK2 include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, and the second antigen-binding domains that bind to lymphocyte antigens include HCDR1 of SEQ ID NOs. 255, HCDR2 of SEQ ID NOs. 256, HCDR3 of SEQ ID NOs. 257, LCDR1 of SEQ ID NOs. 258, LCDR2 of SEQ ID NOs. 259, and LCDR3 of SEQ ID NOs. 261.

[0799] In certain embodiments, the first antigen-binding domains that bind to hK2 include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 194, 195, 172, 173, 174, and 175, respectively, and the second antigen-binding domains that bind to lymphocyte antigens include HCDR1 of SEQ ID NOs. 255, HCDR2 of SEQ ID NOs. 256, HCDR3 of SEQ ID NOs. 257, LCDR1 of SEQ ID NOs. 258, LCDR2 of SEQ ID NOs. 259, and LCDR3 of SEQ ID NOs. 261.

[0800] In certain embodiments, the first antigen-binding domains that bind to hK2 include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, and the second antigen-binding domains that bind to lymphocyte antigens include VH of SEQ ID NOs. 248 and VL of SEQ ID NOs. 251.

[0801] In certain embodiments, the first antigen-binding domains that bind to hK2 include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 194, 195, 172, 173, 174, and 175, respectively, and the second antigen-binding domains that bind to lymphocyte antigens include VH of SEQ ID NOs. 248 and VL of SEQ ID NOs. 251.

[0802] In certain embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second antigen-binding domain that binds to lymphocyte antigens includes HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[0803] In certain embodiments, the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second antigen-binding domain that binds to lymphocyte antigens includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0804] This disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the isolated multispecific protein is as follows: The first binding domain that binds to hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to the lymphocyte antigen includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 275, 258, 259, and 261, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively; The first domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively; The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively; The first binding domain that binds to the hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to the lymphocyte antigen includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively; or The first binding domain that binds to the hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds to the lymphocyte antigen includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively.

[0805] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0806] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 275, 258, 259, and 261, respectively.

[0807] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0808] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively.

[0809] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0810] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0811] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0812] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively.

[0813] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0814] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0815] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0816] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0817] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0818] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0819] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0820] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0821] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0822] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively.

[0823] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0824] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively.

[0825] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0826] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0827] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0828] The disclosure also provides isolated multispecific proteins comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds to a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively.

[0829] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0830] This disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second antigen-binding domain that binds to a lymphocyte antigen, wherein the isolated multispecific protein is as follows: The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 166 and VL of SEQ ID NO: 444, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; The first binding domain that binds to the hK2 comprises VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205, and the second binding domain that binds to the lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; The first binding domain that binds to the hK2 includes VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; or The first binding domain that binds to the hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0831] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0832] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0833] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0834] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[0835] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0836] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0837] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0838] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[0839] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0840] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0841] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0842] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0843] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0844] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 166 and VL of SEQ ID NO: 444, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0845] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0846] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0847] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0848] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[0849] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0850] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[0851] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0852] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0853] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0854] The disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to a lymphocyte antigen, wherein the first binding domain that binds to hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second binding domain that binds to a lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0855] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein. In some embodiments, a first antigen-binding domain that binds to hK2 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding domain that binds to a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

[0856] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include an Fc region.

[0857] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain.

[0858] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH3 domain.

[0859] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include a CH2 domain and a CH3 domain.

[0860] In some embodiments, the fragment of the first Ig steady-state region and / or the fragment of the second Ig steady-state region include at least a portion of the hinge, a CH2 domain, and a CH3 domain.

[0861] In some embodiments, the Ig constant region fragment includes a hinge, a CH2 domain, and a CH3 domain.

[0862] In some embodiments, the multispecific protein further includes a second linker (L2) between a first antigen-binding domain that binds to hK2 and a first Ig constant region or a fragment of the first Ig constant region, and between a second antigen-binding domain that binds to a lymphocyte antigen and a second Ig constant region or a fragment of the second Ig constant region.

[0863] In some embodiments, L2 includes the amino acid sequence of SEQ ID NOs: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

[0864] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1, IgG2, and IgG3 or IgG4 isotypes.

[0865] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1 isotypes.

[0866] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG2 isotypes.

[0867] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG3 isotypes.

[0868] In some embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG4 isotypes.

[0869] In certain embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are IgG1 isotypes.

[0870] The first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region may be further genetically modified as described herein.

[0871] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation that reduces the binding of multispecific proteins to FcγR.

[0872] In some embodiments, at least one mutation that reduces the binding of multispecific proteins to FcγR is found in F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A32 The group is selected from 7G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, and the residue numbering follows the EU index. In certain embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region and / or the second Ig steady-state region or a fragment of the second Ig steady-state region contain the following mutation: L234A_L235A_D265S.

[0873] In some embodiments, the first Ig constant region or fragment of the first Ig constant region, and the second Ig constant region or fragment of the second Ig constant region, include at least one mutation that enhances the binding of the multispecific protein to the Fcγ receptor (FcγR).

[0874] In some embodiments, at least one mutation that enhances the binding of the multispecific protein to FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, where the residue numbering follows the EU index.

[0875] In some embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[0876] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, include at least one mutation that modulates the half-life of the multispecific protein.

[0877] In some embodiments, at least one mutation that modulates the half-life of a multispecific protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, where the residue numbering follows the EU index.

[0878] In some embodiments, the multispecific protein comprises at least one mutation in the CH3 domain of a first Ig constant region or a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of a second Ig constant region or a fragment of the second Ig constant region.

[0879] In some embodiments, at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y4 The residues are selected from the group consisting of 07V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, and the residue numbering follows the EU index. In certain embodiments, the first Ig steady-state region or a fragment of the first Ig steady-state region includes the following mutation: T350V_T366L_K392L_T394W and / or the second Ig steady-state region or a fragment of the second Ig steady-state region includes the following mutation: T350V_L351Y_F405A_Y407V.

[0880] In some embodiments, the first Ig steady region or a fragment of the first Ig steady region and the second Ig steady region or a fragment of the second Ig steady region include the following mutations: L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig steady-state region and L235A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig steady-state region; or L235A_L235A_D265S_T350V_T366L_K392L_T394W in the first steady-state Ig region and L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second steady-state Ig region.

[0881] In some embodiments, the first Ig steady region or a fragment of the first Ig steady region and the second Ig steady region or a fragment of the second Ig steady region include the following mutations: L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig steady-state region and L234A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig steady-state region; or L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first steady-state Ig region and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second steady-state Ig region.

[0882] In some embodiments, the first Ig heavy chain constant region or fragment of the first Ig heavy chain constant region includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 378.

[0883] In certain embodiments, the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region includes an amino acid sequence identical to that of SEQ ID NO: 378.

[0884] In some embodiments, the first Ig light chain constant region or fragment of the first Ig light chain constant region includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 309.

[0885] In certain embodiments, the first Ig light chain constant region or a fragment of the first Ig light chain constant region includes an amino acid sequence identical to that of SEQ ID NO: 309.

[0886] In some embodiments, the second Ig constant region or fragment of the second Ig constant region includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 109.

[0887] In certain embodiments, the second Ig constant region or fragment of the second Ig constant region includes an amino acid sequence identical to that of SEQ ID NO: 109.

[0888] In a particular embodiment, the first Ig heavy chain constant region includes an amino acid sequence identical to that of SEQ ID NO: 378, the first Ig light chain constant region includes an amino acid sequence identical to that of SEQ ID NO: 309, and the second Ig constant region includes an amino acid sequence identical to that of SEQ ID NO: 109.

[0889] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having an amino acid sequence identical to that of SEQ ID NO: 378.

[0890] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having an amino acid sequence identical to that of SEQ ID NO: 309.

[0891] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378, and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309.

[0892] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), and a second Ig constant region or a fragment of a second Ig constant region having an amino acid sequence identical to that of SEQ ID NO: 109.

[0893] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378, and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309, the isolated protein further comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0894] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, and a first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO. 378.

[0895] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having the same amino acid sequence as SEQ ID NO. 309.

[0896] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NOs. 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NOs. 309.

[0897] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), including HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261, and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0898] In certain embodiments, the isolated protein disclosed herein comprises: a first antigen-binding domain that binds to hK2, comprising HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively; (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO. 378; and (ii) a first Ig light chain comprising the same amino acid sequence as SEQ ID NO. 309. The isolated protein comprises a constant region or a fragment of a first Ig light chain constant region, the isolated protein further comprising a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), the second antigen-binding domain that binds to a lymphocyte antigen, comprising HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261, and a second Ig constant region or a fragment of a second Ig constant region having the same amino acid sequence as SEQ ID NO: 109.

[0899] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and a first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region having the same amino acid sequence as SEQ ID NO: 378.

[0900] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and a first Ig light chain constant region or a fragment of the first Ig light chain constant region having the same amino acid sequence as SEQ ID NO: 309.

[0901] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO: 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NO: 309.

[0902] In certain embodiments, the isolated protein disclosed herein comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), comprising the amino acid sequence of SEQ ID NO: 331, and a second Ig constant region or fragment of a second Ig constant region comprising the same amino acid sequence as SEQ ID NO: 109.

[0903] In certain embodiments, the isolated protein disclosed herein comprises a first antigen-binding domain that binds to hK2, comprising VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; (i) a first Ig heavy chain constant region or fragment of a first Ig heavy chain constant region comprising the same amino acid sequence as SEQ ID NO: 378; and (ii) a first Ig light chain constant region or fragment of a first Ig light chain constant region comprising the same amino acid sequence as SEQ ID NO: 309, wherein the isolated protein further comprises a second antigen-binding domain that binds to a lymphocyte antigen (e.g., CD3), comprising the same amino acid sequence as SEQ ID NO: 331; and a second Ig constant region or fragment of a second Ig constant region comprising the same amino acid sequence as SEQ ID NO: 109.

[0904] Production of multispecific proteins containing antigen-binding fragments that bind to hK2 The antigen-binding fragments that bind to hK2 in this disclosure may be genetically modified from multispecific antibodies, and such antibodies are also included within the scope of the present invention.

[0905] The antigen-binding fragment that binds to hK2 may be prepared using Fab arm exchange and genetically engineered into a full-length multispecific antibody in which substitutions are introduced into two monospecific bivalent antibodies within the Ig constant region CH3 domain to facilitate Fab arm exchange in vitro. In this method, two monospecific bivalent antibodies are genetically engineered to have specific substitutions in the CH3 domain to promote heterodimer stability. These antibodies are incubated together under sufficiently reducing conditions for cysteine ​​in the hinge region to isomerize the disulfide bond, thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Typical reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation can be performed at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, for example, pH 7.0 or pH 7.4, for at least 90 minutes.

[0906] Possible CH3 mutation techniques include knob-in-hole mutations (Genentech), electrostatic match mutations (Chugai, Amgen, NovoNordisk, Oncomed), strand-exchange gene-manipulated domain bodies (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

[0907] Knob-in-hole mutations, disclosed for example in International Publication No. 1996 / 027011, include interfacial mutations of the CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in a preferential interaction between the first and second CH3 regions. Exemplary CH3 region mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0908] Heavy chain heterodimer formation can be facilitated by using electrostatic interactions by substituting a positively charged residue on the first CH3 region and a negatively charged residue on the second CH3 region, as described in U.S. Patent Application Publications 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532.

[0909] Other asymmetric mutations that can be used to promote heavy chain heterodimerization include L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T3 described in U.S. Patent Application Publication No. 2012 / 0149876 or 2013 / 0195849 (Zymeworks). These are 66L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0910] SEED body mutations, as described in U.S. Patent Application Publication No. 2007 / 0287170, involve the substitution of selected IgG residues with IgA residues to promote heavy chain heterodimerization.

[0911] Other exemplary variants that may be used include R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366W, and Y349C_T366W / S354C_T366S, as described in International Publication No. 2007 / 147901, International Publication No. 2011 / 143545, International Publication No. 2013 / 157954, International Publication No. 2013 / 096291, and U.S. Patent Application Publication No. 2018 / 0118849. 6S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y349D, L351K / L368E, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E_D282K_K322D / D239K_E240K_K292D, K392D_K409D / D356K_D399K.

[0912] Duobody® variants (Genmab) are disclosed, for example, in U.S. Patent No. 9,150,663 and U.S. Patent Application Publication No. 2014 / 0303356, and include variants such as F405L / K409R, wild-type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[0913] Further bispecific or multispecific structures that can incorporate an antigen-binding fragment that binds to hK2 include: bivariable domain immunoglobulin (DVD) (International Publication No. 2009 / 134776; DVD is a full-length antibody comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure, with the linker being optional), structures containing various dimerization domains for conjugating two antibody arms with different specificities, such as leucine zippers or collagen dimerization domains (International Publication No. 2012 / 022811, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441), two or more domain antibodies (dAb) conjugated together, diabodies, heavy chain-only antibodies such as camelid antibodies and engineered camelid antibodies, dual-target (DT)-Ig (GSK / Domantis), 2in1 antibodies (Genentech), cross-linked Mab (Karmanos Cancer This includes mAb2 (F-Star), CovX body (CovX / Pfizer), IgG-like bispecificity (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec), TvAb (Roche), ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), biaffinity retargeting technology (Fc-DART) (MacroGenics), and bivalent (ScFv)2-Fab (National Research Center for Antibody Medicine--China), biactive or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), divalent bispecificity (Biotecnol), and Fab-Fv (UCB-Celltech).ScFv antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE) (Micromet), tandem diabodies (Tandab) (Affimed), biaffinity retargeting technology (DART) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), bitarget nanobodies (Ablynx), and bitarget heavy chain-only domain antibodies.

[0914] The antigen-binding domains that bind to hK2 in this disclosure may also be genetically engineered into a multispecific protein comprising three polypeptide chains. In such designs, at least one antigen-binding domain is in the form of an scFv. Exemplary designs include the following (where "1" represents the first antigen-binding domain, "2" represents the second antigen-binding domain, and "3" represents the third antigen-binding domain): Design 1: Chain A) scFv1-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3 Design 2: Chain A) scFv1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3 Design 3: Chain A) scFv1-CH1-Hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-Hinge-CH2-CH3 Design 4: Chain A) CH2-CH3-scFv1; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3 As described in U.S. Patent Application Publication No. 2012 / 0149876 or No. 2013 / 0195849 (Zymeworks), variants L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V Genetic manipulation of CH3 such as L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W may be incorporated into designs 1-4.

[0915] In a particular embodiment, the design is as follows: Chain A) scFv-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3.

[0916] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (such as CD3), wherein the first antigen-binding domain that binds to hK2 is HCDR1 of SEQ ID NOs. 63, 72, 141, 147, 170, 176, 188, 194, 196, 198, 200, 206, or 216, and SEQ ID NOs. 64, 65, 73, 142, 148, 171, 177, 188, 189, 19 Includes HCDR2 of sequence numbers 5, 197, 199, 201, 207, or 217; HCDR3 of sequence numbers 66, 143, 172, 178, 184, 190, 208, or 218; LCDR1 of sequence numbers 67, 68, 144, 173, 179, 182, 185, or 191; LCDR2 of sequence numbers 69, 70, 145, 174, 180, 186, 192, or 470; and LCDR3 of sequence numbers 71, 146, 175, 181, 187, 193, or 209.

[0917] In some embodiments, the lymphocyte antigen is CD3. In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence IDs 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0918] In some embodiments, the lymphocyte antigen is CD3. In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, or SEQ ID NOs. 194, 195, 172, 173, 174, and 175, respectively.

[0919] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes: VH of sequence number 137 and VL of sequence number 138; VH of sequence number 162 and VL of sequence number 163; VH of sequence number 164 and VL of sequence number 165; VH of sequence number 166 and VL of sequence number 167; VH of sequence number 168 and VL of sequence number 169; or VH of sequence number 204 and VL of sequence number 205.

[0920] In some embodiments, the lymphocyte antigen is CD3.

[0921] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74.

[0922] In some embodiments, the lymphocyte antigen is CD3. In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0923] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163.

[0924] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, which are at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to VH of SEQ ID NO: 162.

[0925] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0926] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0927] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes the VH of SEQ ID NOs. 4, 5, 6, 137, 139, 159, or 161 and the VL of SEQ ID NOs. 1, 2, 3, 140, or 160, provided that the antigen-binding domain that binds to hK2 does not include both the VH of SEQ ID NOs. 5 and the VL of SEQ ID NOs. 2.

[0928] In some embodiments, the lymphocyte antigen is CD3.

[0929] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 includes: VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160; VH of sequence number 139 and VL of sequence number 1; VH of sequence number 139 and VL of sequence number 2; VH of sequence number 139 and VL of sequence number 3; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 139 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 1; VH of sequence number 159 and VL of sequence number 2; VH of sequence number 159 and VL of sequence number 3; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 161 and VL of sequence number 1; VH of sequence number 161 and VL of sequence number 2; VH of sequence number 161 and VL of sequence number 3; VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or VH of sequence number 161 and VL of sequence number 160.

[0930] In some embodiments, the lymphocyte antigen is CD3.

[0931] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 133, 134, 135, 136, 308, 316, 318, 319, 320, 321, 322, 323, 324, 325, 404, 405, 406, 407, 408, or 409.

[0932] In some embodiments, the lymphocyte antigen is CD3.

[0933] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 contains an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404 or 405.

[0934] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds to hK2 comprises the amino acid sequence of SEQ ID NO: 404 or 405.

[0935] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes HCDR1 of SEQ ID NO: 255 or 116, HCDR2 of SEQ ID NO: 256 or 117, HCDR3 of SEQ ID NO: 257 or 118, or LCDR1 of SEQ ID NO: 258 or 119, LCDR2 of SEQ ID NO: 259 or 120, and LCDR3 of SEQ ID NO: 260, 261, or 121.

[0936] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 157.

[0937] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, the second antigen-binding domain that binds to the lymphocyte antigen comprising: HCDR1 of sequence number 255, HCDR2 of sequence number 256, HCDR3 of sequence number 257, LCDR1 of sequence number 258, LCDR2 of sequence number 259, and LCDR3 of sequence number 260; or HCDR1 (sequence number 255), HCDR2 (sequence number 256), HCDR3 (sequence number 257), LCDR1 (sequence number 258), LCDR2 (sequence number 259), and LCDR3 (sequence number 261).

[0938] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, the second antigen-binding domain that binds to the lymphocyte antigen comprising: VH of sequence number 248 and VL of sequence number 249; VH of sequence number 248 and VL of sequence number 250; VH of sequence number 248 and VL of sequence number 251; VH of sequence number 248 and VL of sequence number 252; VH of sequence number 248 and VL of sequence number 253; or VH of sequence number 248 and VL of sequence number 254.

[0939] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes a VH that is at least 80% (at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 251.

[0940] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[0941] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, the second antigen-binding domain that binds to the lymphocyte antigen comprising: HCDR1 of sequence number 116, HCDR2 of sequence number 117, HCDR3 of sequence number 118, LCDR1 of sequence number 119, LCDR2 of sequence number 120, and LCDR3 of sequence number 121; or VH of sequence number 122 and VL of sequence number 123.

[0942] In certain embodiments, the isolated multispecific protein comprises a first binding domain that binds to hK2 and a second binding domain that binds to a lymphocyte antigen, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, or LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[0943] In some embodiments, the second binding domain of the isolated multispecific protein that binds to the lymphocyte antigen comprises the amino acid sequence of SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, or 337.

[0944] In certain embodiments, the second binding domain of the isolated multispecific protein that binds to the lymphocyte antigen includes an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 331.

[0945] This disclosure also provides an isolated multispecific protein comprising a first domain that binds to hK2 and a second domain that binds to CD3, wherein the isolated multispecific protein is as follows: The first binding domain that binds to the hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 275, 258, 259, and 260, respectively; The first binding domain that binds to the hK2 includes scFV of SEQ ID NO: 136, and the second binding domain that binds to the lymphocyte antigen includes VH of S...

Claims

1. Isolated proteins comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively.

2. The isolated protein according to claim 1, The antigen-binding domain that binds to the aforementioned hK2 is scFv, (scFv) ２ , Fv, Fab, or F(ab') ２ That is, Isolated protein.

3. The isolated protein according to claim 2, The antigen-binding domain that binds to the aforementioned hK2 is Fab, or An isolated protein in which the antigen-binding domain that binds to the aforementioned hK2 is scFV.

4. An isolated protein according to any one of claims 1 to 3, The antigen-binding domain that binds to hK2, i. Includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; or ii. An isolated protein comprising a VH that is at least 90%, at least 95%, at least 99%, or 100% identical to the VH of SEQ ID NO: 162, and a VL that is at least 90%, at least 95%, at least 99%, or 100% identical to the VL of SEQ ID NO:

163.

5. An isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), The following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; b. Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; c. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; d. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; e. Sequence IDs 206, 207, 208, 182, 470, and 209, respectively; f. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; g. Sequence IDs 194, 195, 172, 173, 174, and 175, respectively; h. Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; i. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; j. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or k. Sequence IDs 216, 217, 218, 182, 470, and 209, respectively; Isolated proteins containing these proteins.

6. The isolated protein according to claim 5, The antigen-binding domain that binds to the aforementioned hK2 is scFv, (scFv) ２ , Fv, Fab, or F(ab') ２ That is, Isolated protein.

7. An isolated protein according to claim 5 or 6, The antigen-binding domain that binds to the aforementioned hK2 includes the following: a. VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138; or b. VH of sequence number 164 and VL of sequence number 165; or c. VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167; or d. VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; or e. VH of sequence number 204 and VL of sequence number 205; or f. VH of sequence number 166 and VL of sequence number 444, Isolated protein.

8. Isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively.

9. The isolated protein according to claim 8, a. The antigen-binding domain that binds to hK2 includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140, and / or; b. The antigen-binding domain that binds to the hK2 is scFv, (scFv) ２ , Fv, Fab, or F(ab') ２ This is an isolated protein.

10. An isolated protein according to any one of claims 1 to 4, a. The isolated protein is a single-specific protein; or b. An isolated protein in which the isolated protein is a multispecific protein.

11. The isolated protein according to claim 10b, An isolated protein wherein the multispecific protein is a bispecific protein or a tripspecific protein.

12. An isolated protein according to any one of claims 5 to 9, a. It is a single-specific protein; or b. Isolated proteins that are multispecific proteins.

13. The isolated protein according to claim 12b, An isolated protein containing an antigen-binding domain that binds to antigens on lymphocytes.

14. The isolated protein according to claim 13, Isolated protein wherein the antigen on the lymphocyte is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

15. The isolated protein according to claim 14, The antigen on the lymphocyte is CD3ε, and The antigen-binding domain that binds to the aforementioned CD3ε includes the following: a. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; or b. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; or c. Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, HCDR3 of SEQ ID NO: 118, Light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; or d. VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; or e. VH of sequence number 248 and VL of sequence number 157, Isolated protein.

16. An isolated protein according to any one of claims 1 to 15, An isolated protein that is conjugated to the half-life extension portion.

17. The isolated protein according to claim 16, An isolated protein in which the half-life extension portion is immunoglobulin (Ig), a fragment of Ig, an Ig constant region, a fragment of the Ig constant region, an Fc region, transferrin, albumin, an albumin-binding domain, or polyethylene glycol.

18. The isolated protein according to claim 17, An isolated protein comprising the aforementioned Ig constant region fragment, including the Fc region.

19. The isolated protein according to claim 18, a. The fragment of the Ig constant region includes a CH2 domain; b. The fragment of the Ig constant region includes a CH3 domain; or c. An isolated protein in which the Ig constant region fragment contains a CH2 domain and a CH3 domain.

20. The isolated protein according to claim 19c, An isolated protein in which the Ig constant region fragment comprises at least a portion of the hinge, a CH2 domain, and a CH3 domain.

21. An isolated protein according to any one of claims 17 to 20, The antigen-binding domain that binds to hK2, a. Conjugated to the N-terminus of the Ig steady-state region or a fragment of the Ig steady-state region; b. Conjugated to the C-terminus of the Ig steady-state region or a fragment of the Ig steady-state region; or c. An isolated protein conjugated to the Ig constant region or a fragment of the Ig constant region via a second linker (L2).

22. An isolated protein according to any one of claims 17 to 21, An isolated protein in which the Ig constant region or a fragment of the Ig constant region is an IgG1, IgG2, IgG3, or IgG4 isotype.

23. The isolated protein according to claim 22, An isolated protein in which the Ig constant region or a fragment of the Ig constant region is IgG1.

24. An isolated protein according to any one of claims 17 to 23, An isolated protein wherein the Ig constant region or a fragment of the Ig constant region contains at least one mutation that reduces the binding of the protein to the Fcγ receptor (FcγR).

25. The isolated protein according to claim 24, The at least one mutation that reduces the binding of the protein to the FcγR is F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D Isolated proteins selected from the group consisting of 365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, in which residue numbering follows the EU index.

26. An isolated protein according to any one of claims 17 to 25, An isolated protein in which the Ig constant region or a fragment of the Ig constant region contains at least one mutation that modulates the half-life of the protein.

27. ​​The isolated protein according to claim 26, An isolated protein in which at least one mutation that regulates the half-life of the protein is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, and the residue numbering follows the EU index.

28. An isolated protein according to any one of claims 17 to 27, The isolated protein is one in which the protein contains at least one mutation in the CH3 domain of the Ig constant region.

29. The isolated protein according to claim 28, The at least one mutation in the CH3 domain of the Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407 Isolated proteins selected from the group consisting of V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, in which residue numbering follows the EU index.

30. The isolated protein according to claim 29, An isolated protein in which the mutation in the CH3 domain of the Ig constant region is T350V_T366L_K392L_T394W or T350V_L351Y_F405A_Y407V.

31. An isolated protein according to claim 29 or 30, An isolated protein in which the IgG constant region includes SEQ ID NOs: 109, 110, 158, or 378.

32. It is an isolated protein, a. An isolated protein according to any one of claims 1 to 14, comprising an HC that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 354 and an LC that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 221; or b. The isolated protein according to any one of claims 1 to 7 or 16 to 31, comprising: i. The heavy chain (HC) of SEQ ID NO: 210 and the light chain (LC) of SEQ ID NO: 221; ii. HC of SEQ ID NO: 211 and LC of SEQ ID NO: 222; iii. HC of SEQ ID NO: 212 and LC of SEQ ID NO: 223; iv. HC of SEQ ID NO: 213 and LC of SEQ ID NO: 224; or v. HC of SEQ ID NO: 219 and LC of SEQ ID NO:

220.

33. An isolated protein comprising an antigen-binding domain that binds to hK2, wherein the antigen-binding domain is a. Includes the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively; and / or ii. VH of sequence number 162 and VL of sequence number 163; and / or iii. HC of sequence number 354 and LC of sequence number 221; b. Including the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively; and / or ii. VH of sequence number 162 and VL of sequence number 163; and / or iii. HC of SEQ ID NO: 210 and LC of SEQ ID NO: 221; c. Including the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 176, 177, 178, 179, 180, and 181, respectively; and / or ii. VH of sequence number 164 and VL of sequence number 165; and / or iii. HC of sequence number 211 and LC of sequence number 222; d. Including the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 183, 184, 185, 186, and 187, respectively; and / or ii. VH of sequence number 166 and VL of sequence number 167; and / or iii. HC of SEQ ID NO: 212 and LC of SEQ ID NO: 223; e. Including the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 188, 189, 190, 191, 192, and 193, respectively; and / or ii. VH of sequence number 168 and VL of sequence number 169; and / or iii. HC of SEQ ID NO: 213 and LC of SEQ ID NO: 224; or f. Including the following: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 206, 207, 208, 182, 470, and 209, respectively; and / or ii. VH of sequence number 204 and VL of sequence number 205; and / or iii. HC of sequence number 219 and LC of sequence number 220; Isolated protein.

34. The isolated protein according to claim 33c to 33f, The isolated protein is a multispecific protein containing an antigen-binding domain that binds to CD3ε.

35. It comprises a first antigen-binding domain that binds to hK2 and a second antigen-binding domain that binds to lymphocyte antigens, wherein the first antigen-binding domain that binds to hK2 is a. Each containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of sequence numbers 170, 171, 172, 173, 174, and 175; or b. Including VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, Isolated anti-hK2 / anti-CD3 protein.

36. The isolated anti-hK2 / anti-CD3 protein according to claim 35, a. The first antigen-binding domain that binds to hK2 includes a VH that is at least 90%, at least 95%, at least 99%, or 100% identical to the VH of SEQ ID NO: 162, and / or a VL that is at least 90%, at least 95%, at least 99%, or 100% identical to the VL of SEQ ID NO: 163, and / or b. Including an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 404 or 405, Isolated anti-hK2 / anti-CD3 protein.

37. An isolated anti-hK2 / anti-CD3 protein according to claim 35 or 36, Isolated anti-hK2 / anti-CD3 protein, wherein the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

38. The isolated anti-hK2 / anti-CD3 protein according to claim 37, wherein the lymphocyte antigen is CD3ε.

39. An isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 38, An isolated anti-hK2 / anti-CD3 protein wherein the first antigen-binding domain that binds to hK2 comprises scFv, (scFv)2, Fv, Fab, or F(ab')2, and / or the second antigen-binding domain that binds to the lymphocyte antigen comprises scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

40. The isolated anti-hK2 / anti-CD3 protein according to claim 39, a. The first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen comprises Fab; b. The second antigen-binding domain that binds to the lymphocyte antigen contains VHH; c. The first antigen-binding domain that binds to hK2 and / or the second antigen-binding domain that binds to the lymphocyte antigen includes scFV; or d. An isolated anti-hK2 / anti-CD3 protein in which the first antigen-binding domain that binds to hK2 contains Fab, and the second antigen-binding domain that binds to the lymphocyte antigen contains scFV.

41. The isolated anti-hK2 / anti-CD3 protein according to claim 40, The scFv is an isolated anti-hK2 / anti-CD3 protein comprising VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH), from the N-terminus to the C-terminus.

42. The isolated anti-hK2 / anti-CD3 protein according to claim 41, The above L1 includes the following: A. Approximately 5 to 50 amino acids, B. Approximately 5 to 40 amino acids, C. Approximately 10 to 30 amino acids, or D. Approximately 10 to 20 amino acids, Isolated anti-hK2 / anti-CD3 protein.

43. The isolated anti-hK2 / anti-CD3 protein according to claim 42, Isolated anti-hK2 / anti-CD3 protein in which L1 contains the amino acid sequence of SEQ ID NOs: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

44. comprising a first antigen-binding domain that binds to hK2 and a second antigen-binding domain that binds to a lymphocyte antigen, wherein the first antigen-binding domain that binds to hK2 is a. i. Including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: A. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; B. Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; C. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; D. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; E. Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; F. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; G. Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; H. Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; I. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; J. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or K. Sequence IDs 216, 217, 218, 181, 470, and 209, respectively; or ii. Includes the following: A. VH of sequence number 137 and VL of sequence number 138; B. VH of sequence number 164 and VL of sequence number 165; C. VH of sequence number 166 and VL of sequence number 167; D. VH of sequence number 168 and VL of sequence number 169; E. VH of sequence number 204 and VL of sequence number 205; or F. VH of sequence number 166 and VL of sequence number 444; or b. Including VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140; and / or c. Isolated anti-hK2 / anti-CD3 protein containing the amino acid sequence of SEQ ID NOs: 133, 134, 135, 136, 308, 316, 324, 325, 406, 407, 408, or 409.

45. The isolated anti-hK2 / anti-CD3 protein according to claim 44, Isolated anti-hK2 / anti-CD3 protein, wherein the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

46. An isolated anti-hK2 / anti-CD3 protein according to claim 44 or 45, An isolated anti-hK2 / anti-CD3 protein wherein the first antigen-binding domain that binds to hK2 comprises scFv, (scFv)2, Fv, Fab, or F(ab')2, and / or the second antigen-binding domain that binds to the lymphocyte antigen comprises scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

47. An isolated anti-hK2 / anti-CD3 protein according to any one of claims 44 to 46, a. The second antigen-binding domain that binds to the lymphocyte antigen is i. Including HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; ii. Includes the following: A. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; or B. VH of sequence number 248 and VL of sequence number 251; and / or iii. A VH that is at least 90%, at least 95%, at least 99%, or 100% identical to VH of SEQ ID NO: 248 and a VL that is at least 90%, at least 95%, at least 99%, or 100% identical to VL of SEQ ID NO: 251; or b. The second antigen-binding domain that binds to the lymphocyte antigen includes the following: i. HCDR1 of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, HCDR3 of SEQ ID NO: 118, LCDR1 of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; ii. VH of sequence number 122 and VL of sequence number 123; iii. Containing the amino acid sequence of SEQ ID NO: 331; or iv. An isolated anti-hK2 / anti-CD3 protein containing an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

331.

48. An isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 47, An isolated anti-hK2 / anti-CD3 protein, wherein a first antigen-binding domain that binds to hK2 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or a second antigen-binding domain that binds to the lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

49. The isolated anti-hK2 / anti-CD3 protein according to claim 48, The isolated anti-hK2 / anti-CD3 protein comprises a fragment of the Ig constant region containing an Fc region.

50. The isolated anti-hK2 / anti-CD3 protein according to claim 49, a. The fragment of the Ig constant region includes a CH2 domain, b. The fragment of the Ig constant region contains a CH3 domain, or c. An isolated anti-hK2 / anti-CD3 protein comprising the Ig constant region fragment containing a CH2 domain and a CH3 domain.

51. The isolated anti-hK2 / anti-CD3 protein according to claim 50c, An isolated anti-hK2 / anti-CD3 protein in which the Ig constant region fragment comprises at least a portion of the hinge, a CH2 domain, and a CH3 domain.

52. The isolated anti-hK2 / anti-CD3 protein according to claim 49, An isolated anti-hK2 / anti-CD3 protein wherein the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region are IgG1, IgG2, IgG3, or IgG4 isotypes.

53. The isolated anti-hK2 / anti-CD3 protein according to claim 52, The first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region are a. Being IgG1; and / or b. An isolated anti-hK2 / anti-CD3 protein containing at least one mutation that reduces the binding of the multispecific protein to FcγR.

54. The isolated anti-hK2 / anti-CD3 protein according to claim 53b, The at least one mutation that reduces the binding of the multispecific protein to the FcγR is F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D36 Isolated anti-hK2 / anti-CD3 proteins selected from the group consisting of 5E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 deletion / G237A / P238S, with residue numbering following the EU index.

55. The isolated anti-hK2 / anti-CD3 protein according to claim 52, An isolated anti-hK2 / anti-CD3 protein wherein the first Ig constant region or a fragment of the first Ig constant region, and the second Ig constant region or a fragment of the second Ig constant region, contain at least one mutation that modulates the half-life of the multispecific protein.

56. The isolated anti-hK2 / anti-CD3 protein according to claim 55, The isolated anti-hK2 / anti-CD3 protein comprises at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of a fragment of the first Ig constant region and / or at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of a fragment of the second Ig constant region.

57. The isolated anti-hK2 / anti-CD3 protein according to claim 56, The at least one mutation in the CH3 domain of the first Ig constant region or the CH3 domain of the fragment of the first Ig constant region and / or the at least one mutation in the CH3 domain of the second Ig constant region or the CH3 domain of the fragment of the second Ig constant region is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y Isolated anti-hK2 / anti-CD3 proteins selected from the group consisting of / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, with residue numbering following the EU index.

58. An isolated anti-hK2 / anti-CD3 protein according to any one of claims 48 to 57, The first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region or a fragment of the second Ig steady-state region include the following mutations: a. L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig steady-state region and L234A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig steady-state region; or b. L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig steady-state region and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig steady-state region, Isolated anti-hK2 / anti-CD3 protein.

59. The isolated anti-hK2 / anti-CD3 protein according to claim 58, a. The first Ig steady-state region has sequence number 110 or sequence number 378; b. The second Ig steady-state region has SEQ ID NO: 109 or SEQ ID NO: 158; and / or c. An isolated anti-hK2 / anti-CD3 protein, wherein the first Ig constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 378, the second Ig constant region comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 109, and the isolated protein further comprises a light chain constant region comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

309.

60. An isolated anti-hK2 / anti-CD3 protein comprising a first domain that binds to hK2 and a second domain that binds to CD3, a. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 275, 258, 259, and 261, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 136, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 351, HC2 of SEQ ID NO: 358, and LC2 of SEQ ID NO: 267; b. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 136, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 351, HC2 of SEQ ID NO: 359, and LC2 of SEQ ID NO: 272; c. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 134, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 352, HC2 of SEQ ID NO: 358, and LC2 of SEQ ID NO: 267; d. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 134, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 352, HC2 of SEQ ID NO: 359, and LC2 of SEQ ID NO: 272; e. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 325, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 353, HC2 of SEQ ID NO: 358, and LC2 of SEQ ID NO: 267; f. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively; ii. The first antigen-binding domain that binds to hK2 comprises VH of SEQ ID NO: 166 and VL of SEQ ID NO: 444, and the second antigen-binding domain that binds to the lymphocyte antigen comprises scFv of SEQ ID NO: 331; and / or iii. The isolated multispecific protein comprises HC1 of SEQ ID NO: 355, LC1 of SEQ ID NO: 445, and HC2 of SEQ ID NO: 360; g. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively; ii. The first binding domain that binds to hK2 includes VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165, and the second binding domain that binds to the lymphocyte antigen includes scFv of SEQ ID NO: 331; and / or iii. The isolated multispecific protein comprises HC1 of SEQ ID NO: 356, LC1 of SEQ ID NO: 222, and HC2 of SEQ ID NO: 360; h. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 325, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 353, HC2 of SEQ ID NO: 359, and LC2 of SEQ ID NO: 272; i. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 116, 117, 118, 119, 120, and 121, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 316, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 357, HC2 of SEQ ID NO: 359, and LC2 of SEQ ID NO: 272; j. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively; ii. The first binding domain that binds to hK2 includes scFv of SEQ ID NO: 316, and the second binding domain that binds to the lymphocyte antigen includes VH of SEQ ID NO: 248 and VL of SEQ ID NO: 151; and / or iii. The isolated multispecific protein comprises HC1 of SEQ ID NO: 357, HC2 of SEQ ID NO: 358, and LC2 of SEQ ID NO: 267; or k. i. The first binding domain that binds to hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds to the lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs. 255, 256, 257, 258, 259, and 261, respectively; ii. The first antigen-binding domain that binds to hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second antigen-binding domain that binds to the lymphocyte antigen comprises scFv of SEQ ID NO: 331; and / or iii. The isolated multispecific protein includes HC1 of SEQ ID NO: 361, LC1 of SEQ ID NO: 221, and HC2 of SEQ ID NO:

362. Isolated anti-hK2 / anti-CD3 protein.

61. a. An immunoconjugate comprising an isolated protein according to any one of claims 1 to 34, conjugated to a therapeutic agent or contrast agent; or b. An immunoconjugate comprising an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60, conjugated to a therapeutic agent or contrast agent.

62. A pharmaceutical composition, a. comprising an isolated protein according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier; or b. A pharmaceutical composition comprising an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60 and a pharmaceutically acceptable carrier.

63. a. A kit comprising an isolated protein according to any one of claims 1 to 34, an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60, an immunoconjugate according to claim 61a or 61b, or a pharmaceutical composition according to claim 62a or 62b; b. A polynucleotide encoding an isolated protein according to any one of claims 1 to 34; or c. Polynucleotides, A polynucleotide encoding an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60.

64. a. comprising the polynucleotide described in claim 63b; or b. A vector comprising the polynucleotide described in claim 63c.

65. a. comprising the vector described in claim 64a; or b. A host cell comprising the vector described in claim 64b.

66. a. A method for producing an isolated protein according to any one of claims 1 to 34, comprising: culturing a host cell according to claim 65a under conditions in which the protein is expressed; and recovering the protein produced by the host cell; or b. A method for producing an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60, comprising: culturing a host cell according to claim 65b under conditions in which the anti-hK2 / anti-CD3 protein is expressed; and recovering the anti-hK2 / anti-CD3 protein produced by the host cell.

67. A pharmaceutical composition comprising an isolated protein according to any one of claims 1 to 34, an isolated anti-hK2 / anti-CD3 protein according to any one of claims 35 to 60, an immunoconjugate according to claim 61a or 61b, or a pharmaceutical composition according to claim 62a or 62b, a. For treating hK2-expressing cancers in the target population; b. To reduce the amount of hK2-expressing tumor cells in the target population; c. To prevent the establishment of hK2-expressing cancer in the target population; or d. A pharmaceutical composition for treating a non-cancerous condition in subjects at risk of developing hK2-expressing cancer.

68. A pharmaceutical composition according to claim 67, A pharmaceutical composition wherein the hK2-expressing cancer is prostate cancer or breast cancer.

69. The pharmaceutical composition according to claim 68, a. The prostate cancer is recurrent, refractory, malignant, or castration-resistant, or any combination thereof; b. The breast cancer is androgen receptor (AR) positive breast cancer, recurrent, refractory, or malignant breast cancer, or any combination thereof; or c. The non-cancerous condition is characterized by high prostate-specific antigen (PSA) levels in the absence of benign prostatic hyperplasia, benign prostatic hyperplasia (BPH), or diagnosed prostate cancer; and / or d. A pharmaceutical composition in which the pharmaceutical composition is administered in combination with a second therapeutic agent.

70. The pharmaceutical composition according to claim 69d, A pharmaceutical composition in which the second therapeutic agent is surgery, chemotherapy, androgen depletion therapy, or radiation, or any combination thereof.

71. A pharmaceutical composition comprising an immunoconjugate according to claim 61a or 61b for use in a method for detecting the presence of prostate cancer or breast cancer in a subject in vivo, wherein the method comprises administering the immunoconjugate to a subject suspected of having prostate cancer or breast cancer, and visualizing the biological structure to which the immunoconjugate is bound, thereby detecting the presence of prostate cancer or breast cancer.

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