CAIX-targeted single-domain antibodies, bispecific antibodies, antibody-drug conjugates, and use thereof

Abstract

Provided are a class of single-domain antibodies specifically binding to CAIX. The CAIX single-domain antibodies only bind to human CAIX, do not bind to human CAXII or CAXIV, and have relatively high specificity. Further provided are CAIX / CD70 bispecific antibodies prepared on the basis of the single-domain antibodies, and CAIX / CD70 bispecific antibody-drug conjugates, which have significant inhibitory effects on tumors.

Description

Single-domain antibodies, bispecific antibodies, antibody-drug conjugates targeting CAIX and their applications Technical Field

[0001] This invention relates to the field of biology, and in particular to anti-CAIX single-domain antibodies, bispecific antibodies, antibody-drug conjugates, and their applications. Background Technology

[0002] Renal cell carcinoma (RCC) accounts for approximately 2% of cancer diagnoses and deaths worldwide, while clear cell renal cell carcinoma (ccRCC) accounts for 80% of RCC cases. Currently, the first-line standard treatment is immunotherapy. In the CheckMate-214 clinical trial (NCT02231749), the objective response rate (ORR) of nivolumab combined with ipilimumab in patients with advanced renal cell carcinoma was 42%, the median overall survival (OS) was 46.7 months, and the 12-month overall survival rate was 80% (Motzer, et al. (2018) N Engl J Med. 378:1277–90.). Although the 5-year survival rate for early-stage ccRCC is 75%, it drops to 10% for metastatic ccRCC (https: / / seer.cancer.gov / statfacts / html / kidrp.html).

[0003] Studies have found that over 90% of clear nephrotic tumors are positive for carbonic anhydrase IX (CAIX) (Baniak, et al. (2020) Histopathology 77:659-66). Carbonic anhydrase (CA) is a zinc-containing metalloprotein involved in the transfer of CO2 and protons across biological membranes, including intercellular, intracellular, and extracellular spaces (Becker (2020) Br J Cancer 122:157-67). Sixteen CA subtypes have been characterized in mammals, among which carbonic anhydrase IX (CAIX or CAIX) is one of the most active CAs in the CO2 hydration reaction. It is a dimeric transmembrane glycoprotein specifically overexpressed on the surface of hypoxic tumor cells, and its dimerization is mediated by intermolecular disulfide bonds formed between the two monomeric CA domains. CAIX is composed of 459 amino acids, including an N-terminal proteoglycan-like domain (PG), a CA catalytic domain (CA), a single-helix transmembrane region (TM), and an intracellular C-terminal domain (CT). CAIX is a key hypoxia-induced marker, expressed in very limited healthy tissues, but specifically overexpressed on the surface of the vast majority of hypoxic solid tumor cells. Many tumors, including renal cell carcinoma, breast cancer, lung cancer, liver cancer, and colorectal cancer, express high levels of CAIX under hypoxic conditions. Hypoxia also induces CAIX expression in breast cancer cells, enhancing glucose glycolysis and leading to extracellular acidity (Baniak, et al. (2020) Histopathology 77:659-66; Betof, et al. (2012) Br J Cancer 106:916-22; Kon-no, et al. (2006) Lung Cancer 54:409-18; Huang, et al. (2015) PLoS ONE 10:e0119181; (et al. (2016) World J Gastroenterol 22:8168-77). In contrast, CAIX expression in normal tissues is negligible, except in the stomach and gallbladder. CAIX catalyzes CO2, thereby producing protons (H2O). + ) and bicarbonate (HCO3) -CAIX expression is one of the important reasons why solid tumors maintain intracellular pH homeostasis under hypoxic conditions. CAIX expression is associated with drug resistance in cancer cells and also with shorter patient survival (Kim, et al. (2004) Clin Cancer Res, 10:7925–33; Betof, et al. (2012) Br J Cancer 106:916-22). Due to its high expression in tumor cells and its role in the tumor microenvironment, CAIX is considered a potential target for tumor imaging and therapy.

[0004] Drug development targeting CAIX has been ongoing, with over 30 therapeutic projects currently in development, most of which are in preclinical stages. Girentuximab monoclonal antibody was terminated in Phase 3 clinical trials due to insufficient efficacy, and the CAIX-targeting ADC drug BAY-794620 was terminated due to high toxicity. This demonstrates that increasing drug specificity and reducing toxicity are crucial for the successful development of ADC drugs. Bispecific antibody ADCs can reduce toxicity by differentially expressing two targets in normal tissues, thereby reducing their killing effect on normal tissues and increasing the therapeutic window, ultimately achieving better therapeutic effects.

[0005] CD70 is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily, and is typically expressed on activated T cells, B cells, and mature dendritic cells (Zang, et al. (2024) Annu Rev Med). It is the only ligand for CD27 and is involved in regulating the activity of T cells and B cells. Interestingly, CD70 is transiently expressed and is limited to activated cells in healthy individuals. However, CD70 is abnormally expressed in various tumor cells, including renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, lymphoma, pancreatic cancer, and glioblastoma. More than 80% of clear cell renal cell carcinomas overexpress CD70 (Flieswasser, et al. (2019) Cancer (Basel) 11:1611; Law, et al. (2006) Cancer Res 66:2328-37; Jacobs, et al. (2015) Oncotarget. 6:13462–75; Meulenaere, et al. (2016) Pathobiology 83:327-33; Ryan, et al. (2010) Br J Cancer 103:676–84). CD70 expression is associated with tumor immune escape and progression (Wischhusen, et al. (2002) Cancer Res 62:2592-9). Furthermore, CD70 is almost not expressed in normal tissues such as the stomach and gallbladder (https: / / www.proteinatlas.org / ). Therefore, CD70 is also an attractive target for ADC therapy. Based on the high expression of CAIX and CD70 in ccRCC and the differential and relatively specific expression profiles in normal tissues, anti-CAIX / CD70 bispecific ADCs are potential therapeutic agents for ccRCC. Summary of the Invention

[0006] This application utilizes the characteristics of single-domain antibodies (sdAbs) composed of the variable regions of natural heavy chain antibodies lacking the light chain in camel (camel, llama, alpaca and their close relatives) to screen out multiple CAIX single-domain antibodies that specifically target CAIX and have cross-binding with human, monkey and mouse CAIX. Based on this, and combined with other targets, bispecific antibodies and their antibody-drug conjugates were further developed, which have good specificity and better antitumor efficacy.

[0007] A first aspect of this invention provides an anti-CAIX single-domain antibody. The anti-CAIX single-domain antibody comprises CDR1 to CDR3 as shown below:

[0008] The amino acid sequence is as shown in SEQ ID NO.1 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.2 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.3 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3; or

[0009] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0010] The amino acid sequence of the anti-CAIX single-domain antibody provided by the present invention includes an amino acid sequence as shown in any one of SEQ ID NO. 7 to 21 or an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with any one of SEQ ID NO. 7 to 21.

[0011] A second aspect of the present invention provides a fusion protein comprising a first domain, which is an anti-CAIX single-domain antibody as described above; and a second domain having the effect of prolonging the in vivo half-life and / or having the effect of binding to effector cells.

[0012] In some embodiments, the second domain includes one or more of a serum albumin fragment, a polyethylene glycol fragment, and a nanobody that binds HSA; and / or, the second domain includes an immunoglobulin Fc region; and / or, the second domain includes a molecule that has affinity for CD3 present on T cells and / or is capable of binding to CD3 present on T cells.

[0013] In some embodiments, the immunoglobulin Fc region is the human immunoglobulin Fc region, the immunoglobulin is selected from one or more combinations of IgG, IgGA1, IgGA2, IgD, IgE, and IgM, and the IgG is selected from one or more combinations of IgG1, IgG2, IgG3, or IgG4 subtypes.

[0014] A third aspect of the present invention provides a bispecific antibody comprising the anti-CAIX single-domain antibody described in the first aspect of the present invention.

[0015] In embodiments of the present invention, the bispecific antibody further comprises a domain targeting tumor antigens such as EGFR, MET, HER3, HER2, CD70, or TROP2. In some specific embodiments, the bispecific antibody comprises a domain targeting CD70. The CD70-targeting domain is selected from antibody or antigen-binding fragments (e.g., full-length antibody, VHH, Fab, Fab', scFv, Fv, etc.), CD27 protein (e.g., full-length or extracellular domain of CD27), etc.

[0016] CD70 is a type II transmembrane glycoprotein that is aberrantly expressed in various tumor cells, including renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, lymphoma, pancreatic cancer, and glioblastoma. It is overexpressed in over 80% of clear cell renal cell carcinomas, but is almost absent in normal tissues such as the stomach and gallbladder. Based on the high expression of CAIX and CD70 in ccRCC and the differential and relatively specific expression profiles in normal tissues, anti-CAIX / CD70 bispecific anti-ADCs are potential therapeutic agents for ccRCC.

[0017] In some specific embodiments, the CD70-targeting domain is an anti-CD70 antibody or its antigen-binding fragment. The anti-CD70 antibody or its antigen-binding fragment comprises HCDR1 (as shown in SEQ ID NO. 67), HCDR2 (as shown in SEQ ID NO. 68), HCDR3 (as shown in SEQ ID NO. 69), LCDR1 (as shown in SEQ ID NO. 70), LCDR2 (as shown in SEQ ID NO. 71), and LCDR3 (as shown in SEQ ID NO. 72). The amino acid sequence of SEQ ID NO. 71 is LAS.

[0018] In embodiments of the present invention, the anti-CD70 antibody or its antigen-binding fragment comprises a VH amino acid sequence as shown in SEQ ID NO. 73 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity therewith, and / or a VL amino acid sequence as shown in SEQ ID NO. 74 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity therewith. Preferably, the anti-CD70 antibody or its antigen-binding fragment comprises the VH and VL of Vorsetuzumab.

[0019] In embodiments of the present invention, the bispecific antibody further comprises an Fc fragment, wherein the Fc fragment is selected from one or more combinations of IgG, IgA1, IgA2, IgD, IgE, and IgM; in some embodiments, the Fc fragment is selected from the Fc of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the amino acid sequence of the IgG1 Fc has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 75.

[0020] In some embodiments, the bispecific antibody has a left-right asymmetric 1+1 valent structure. In some embodiments, the bispecific antibody consists of three chains, comprising a light chain, a first heavy chain, and a second heavy chain. The first heavy chain comprises an anti-CAIX single-domain antibody fused to an Fc fragment. The light chain and / or the second heavy chain comprises an anti-CD70 antibody or an antigen-binding fragment thereof.

[0021] In some implementations, to facilitate the construction of heterodimers, corresponding Knob and Hole mutations are introduced into the heavy chain Fc region using KIH technology (Shatz, et al. (2013) Mabs 5:872-81). KIH technology is described in US5731168, US7695936, etc. For example, one Fc region contains the amino acid substitution T366W (Knob mutation), and another Fc region contains the amino acid substitution T366S / L368A / Y407V (Hole mutation) (numbered according to EU index). Alternatively, for example, one Fc region contains the amino acid substitution S354C / T366W (Knob mutation), and another Fc region contains the amino acid substitution Y349C / T366S / L368A / Y407V (Hole mutation) (numbered according to EU index).

[0022] In some embodiments, the Fc segment of the first heavy chain contains a Knob mutation and the Fc segment of the second heavy chain contains a Hole mutation, or the Fc segment of the first heavy chain contains a Hole mutation and the Fc segment of the second heavy chain contains a Knob mutation.

[0023] The Fc fragment of the first heavy chain and / or the second heavy chain may also contain amino acid substitutions or mutations for extending the half-life. In some embodiments, the mutation may be selected from the M428L mutation (numbered according to the EU index).

[0024] In some embodiments, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID No. 76 to 83.

[0025] In some specific embodiments of the present invention, the bispecific antibody is a CAIX / CD70 bispecific antibody, wherein the anti-CAIX single-domain antibody comprises:

[0026] The amino acid sequence is as shown in SEQ ID NO.1 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.2 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.3 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3; or

[0027] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0028] The anti-CD70 antibody or its antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO. 67, HCDR2 as shown in SEQ ID NO. 68, HCDR3 as shown in SEQ ID NO. 69, LCDR1 as shown in SEQ ID NO. 70, LCDR2 as shown in SEQ ID NO. 71, and LCDR3 as shown in SEQ ID NO. 72.

[0029] In some specific embodiments of the present invention, the first heavy chain comprises an amino acid sequence as shown in any one of SEQ ID No. 86 to 88, the second heavy chain comprises an amino acid sequence as shown in any one of SEQ ID No. 89 to 90, and the light chain comprises an amino acid sequence as shown in SEQ ID No. 66.

[0030] In some specific embodiments of the present invention, the bispecific antibody comprises:

[0031] The first heavy chain shown in SEQ ID No. 86, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0032] The first heavy chain shown in SEQ ID No. 87, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0033] The first heavy chain shown in SEQ ID No. 88, the second heavy chain shown in SEQ ID No. 90, and the light chain shown in SEQ ID No. 66.

[0034] The present invention further provides an isolated polynucleotide encoding the anti-CAIX single-domain antibody described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, or the bispecific antibody described in the third aspect of the present invention.

[0035] The present invention further provides an expression vector containing the above-isolated polynucleotides.

[0036] The present invention further provides an expression system containing the above-described expression vector or a genome in which exogenous polynucleotides, such as those described above, are integrated.

[0037] The present invention further provides a method for preparing the above-mentioned anti-CAIX single-domain antibody, fusion protein or bispecific antibody, comprising the following steps: culturing the above-mentioned expression system under conditions suitable for expressing the single-domain antibody, fusion protein or bispecific antibody, thereby expressing the single-domain antibody, fusion protein or bispecific antibody, and purifying and separating the single-domain antibody, fusion protein or bispecific antibody.

[0038] A fourth aspect of this invention provides an immunoconjugate or immunodrug conjugate. The immunoconjugate or immunodrug conjugate comprises the anti-CAIX single-domain antibody described in the first aspect of this invention, the fusion protein described in the second aspect of this invention, or the bispecific antibody described in the third aspect of this invention. The immunoconjugate or immunodrug conjugate further comprises one or more of the following: a cytotoxic drug, a molecular gel, a PROTAC molecule, an immune agonist, and a radioactive isotope; preferably, the cytotoxic drug includes, but is not limited to, a tubulin inhibitor, a topoisomerase inhibitor, or a toxin for binding DNA; the molecular gel includes, but is not limited to, pomalidomide, lenalidomide, thalidomide, and GSPT1 degrading agents; the PROTAC molecule includes, but is not limited to, ARV-110, ARV-766, ARV-471, NX-2127, NX-5948, CFT1946, CFT8919, and DT2216; and the radioactive isotope includes, but is not limited to, [missing information - likely a specific type of radioactive isotope]. 18 F, 44 Sc、 47 Sc、 64 Cu、 67 Ga、 86 Y、 89 Zr、90 Y、 99m Tc, 111 In.

[0039] In some preferred embodiments, the immunoconjugate or immunodrug conjugate according to the present invention is an antibody-drug conjugate.

[0040] In some embodiments, the present invention provides an antibody-drug conjugate having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: Ab-(LD) n , formula (I);

[0041] Where Ab is the aforementioned anti-CAIX single-domain antibody or the aforementioned bispecific antibody; L is the linker arm; D is the cytotoxic drug; and n is a number between 1 and 20.

[0042] In some embodiments, the cytotoxic drug D is selected from one or more of the following: maytansine derivative DM1, maytansine derivative DM4, monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), duocarmycin, pyrrolobenzodiazepine (PBD), camptothecin and its derivatives [including but not limited to SN38, eczetine (Exatecan, abbreviated as Exd), Dxd], tubulysins, amanitin, PNU-159682, and calicheamicins.

[0043] In some embodiments, the linker arm L is either a non-cleavable linker arm or a cleavable linker arm. The cleavable linker arm, depending on the cleavage mechanism, includes enzyme-cleavable linkers (e.g., cathepsins, phosphatases, (aryl)sulfatases, β-galactosidases, β-glucosidases, and nitroreductases), acid-cleavable linkers, linkers that cleave under reducing conditions (disulfides, etc., where disulfide bonds break under reducing conditions), and linkers that cleave under exogenous stimuli (infrared sensitive, ultraviolet sensitive, etc.).

[0044] In some embodiments, the linker arm L is carbon-based, amino-based, amide-based, acyl-based, or -(PEG). m -、-(CH2) m - Containing heteroatoms -(CH2) m-、-(C≡C)-、-(CH=CH)-、-O-、-S-、maleimide (Mal or MA), maleimide caproyl (MC), maleimide propionyl (maleimide Propoyl (abbreviated as MP), VA (-Val-Ala-), VC (-Val-Cit-), VK (-Vla-Lys-), AF (-Ala-Phe-), GGFG (-Gly-Gly-Phe-Gly-), p-aminobenzyloxycarbonyl (PAB), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylic acid ester (SMCC), N-succinimide-4-(2-pyridinylthio)valerate (SPP), polyethylene glycol (PEG), pyrimidine, pyridine (py), methanesulfonylpyrimidinyl, methanesulfonylpyridinyl, and one or more combinations thereof, wherein each m is independently selected from an integer from 1 to 20, for example from an integer from 1 to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8. The various groups that make up the linker arm L are connected by chemical bonds. Those skilled in the art should understand that whether or not each of the listed groups has a "-" at both ends does not affect the actual structure of the linker arm L.

[0045] In some implementations, the connecting arm L includes one or more combinations of the following structures:

[0046] Each q is independently selected from integers from 1 to 20, for example, integers from 1 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0047] X is selected from -NH-, -O-, and -S-;

[0048] Su are each independently selected from pentose, penturonic acid, hexose, and hexuronic acid;

[0049] R c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups.

[0050] In some embodiments, the linker arm L is -MC-VC-PAB- and the cytotoxic drug D is MMAE; or the linker arm L is -MC-GGFG- and the cytotoxic drug D is Dxd; or the linker arm L is -pym-PEG8-VA-PAB- and the cytotoxic drug D is Exatecan. Attached Figure Description

[0051] Figure 1A shows the binding curves of the Anti-CAIX single-domain antibody Fc fusion protein anti-CAIX-3H4-Fc and the recombinant human CAIX (137-390) protein;

[0052] Figure 1B shows the binding curves of the Anti-CAIX single-domain antibody Fc fusion protein anti-CAIX-13E6 and the recombinant human CAIX (137-390) protein;

[0053] Figure 2 shows the inhibitory effect of the Anti-CAIX single-domain antibody Fc fusion protein on the catalytic activity of human CAIX.

[0054] Figure 3 shows the flow cytometry binding curves of humanized Anti-CAIX-hu3H4-Fc to CHOK1Q cells overexpressing human CAIX;

[0055] Figure 4 shows the ELISA binding curves of the humanized Anti-CAIX-hu13E6V2-Fc derivative and the recombinant human CAIX (137-390) protein;

[0056] Figure 5 shows the binding curves of the humanized Anti-CAIX-hu13E6V2-Fc derivative to CHOK1Q cells overexpressing human CAIX;

[0057] Figure 6 shows the endocytic activity assay of the humanized Anti-CAIX-VHH-Fc;

[0058] Figure 7A shows the pH-biased binding curves of Anti-CAIX antibody to HT-29 cells;

[0059] Figure 7B shows the pH-biased binding of the Anti-CAIX control antibody CA9hu-1 to human CAIX-overexpressing CHOK1Q cells;

[0060] Figure 8 is a schematic diagram of the anti-CAIX / CD70 dual antibody structure;

[0061] Figure 9A shows the binding activity of DR31503, DR31506, Girentuximab, aCAIX-hu13E6-2D1V2-Fc, and Vorsetumumab with human CAIX-overexpressing CHOK1Q cells.

[0062] Figure 9B shows the binding activity of DR31506 and DR31507 with CHOK1Q cells overexpressing human CAIX;

[0063] Figure 10A shows the binding activity of DR31503, DR31506, Girentuximab, aCAIX-hu13E6V2-2D1-Fc, and Vorsetumumab with human CD70-overexpressing CHOK1Q cells.

[0064] Figure 10B shows the binding activity of DR31506 and DR31507 to human CD70-overexpressing CHOK1Q cells;

[0065] Figure 11A shows the binding activity of DR31503, DR31506, Girentuximab, aCAIX-hu13E6V2-2D1-Fc and Vorsetumumab with 786-O-CAIX cells;

[0066] Figure 11B shows the binding activity of DR31506 and DR31507 to 786-O-CAIX cells;

[0067] Figure 12A shows the serum stability of the CAIX / CD70 bispecific antibody binding activity to CD70;

[0068] Figure 12B shows the serum stability of the CAIX / CD70 bispecific antibody binding activity to CAIX;

[0069] Figure 13A shows the SEC-HPLC detection results of 31506-CPD3;

[0070] Figure 13B shows the RP-HPLC detection results of 31506-CPD3;

[0071] Figure 14A shows the SEC-HPLC detection results of 31503-CPD3;

[0072] Figure 14B shows the RP-HPLC detection results of 31503-CPD3;

[0073] Figure 15A shows the SEC-HPLC detection results of 31507-CPD3;

[0074] Figure 15B shows the RP-HPLC detection results of 31507-CPD3;

[0075] Figure 16A shows the SEC-HPLC detection results of 31506-Dxd;

[0076] Figure 16B shows the RP-HPLC detection results of 31506-Dxd;

[0077] Figure 17A shows the SEC-HPLC detection results of 31503-Dxd;

[0078] Figure 17B shows the RP-HPLC detection results of 31503-Dxd;

[0079] Figure 18A shows the SEC-HPLC detection results of 31506-VCMMAE;

[0080] Figure 18B shows the HIC detection results for 31506-VCMMAE;

[0081] Figure 19A shows the SEC-HPLC detection results of 31503-VCMMAE;

[0082] Figure 19B shows the HIC detection results for 31503-VCMMAE;

[0083] Figure 20 shows the in vitro killing activity of the bispecific antibody ADC conjugated with MMAE against OSRC-2-CAIX cells;

[0084] Figure 21 shows the in vitro killing activity of the bispecific antibody ADC conjugated with Dxd against OSRC-2-CAIX cells;

[0085] Figure 22 shows the in vitro killing activity of the bispecific antibody ADC conjugated with Exatecan against OSRC-2-CAIX cells;

[0086] Figure 23A shows the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, Vorsetumumab-CPD3 and DR31506 isotype 1-CPD3+isotype 2-CPD3 combo against 786-O cells.

[0087] Figure 23B shows the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, Vorsetumumab-CPD3, and DR31506 isotype 1-CPD3+isotype 2-CPD3 combo against 786-O-CAIX cells.

[0088] Figure 24A shows the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, and Vorsetumumab-CPD3 against OSRC-2 cells.

[0089] Figure 24B shows the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, and Vorsetumumab-CPD3 against OSRC-2-CAIX cells.

[0090] Figure 25 shows the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, and Vorsetumumab-CPD3 against HT-29-CAIX cells.

[0091] Figure 26 shows the tumor growth curves of different antibody-drug conjugates in the OS-RC-2-CAIX model;

[0092] Figure 27 shows the tumor inhibition curve of DR31506-CPD3 on the OS-RC-2-CAIX tumor pharmacological model;

[0093] Figure 28 shows the antitumor effect of the antibody-drug conjugate HT-29-CAIX in a tumor pharmacological model.

[0094] Figure 29A shows the antitumor effect of the antibody-drug conjugate in the 786-O-CAIX tumor-bearing model;

[0095] Figure 29B shows the changes in body weight of the antibody-drug conjugate in the 786-O-CAIX tumor-bearing model;

[0096] Figure 30A shows the antitumor effect of the antibody-drug conjugate in the 786-O-CAIX tumor-bearing model;

[0097] Figure 30B shows the changes in body weight of the antibody-drug conjugate in the 786-O-CAIX tumor-bearing model;

[0098] Figure 31A shows the antitumor effect of the antibody-drug conjugate in the PDX model of renal cell carcinoma;

[0099] Figure 31B shows the weight changes of the antibody-drug conjugate in the PDX model of renal cell carcinoma;

[0100] Figure 32A shows the antitumor effect of antibody-drug conjugates in a PDX model of liver cancer;

[0101] Figure 32B shows the weight changes of the antibody-drug conjugate in the PDX model of liver cancer;

[0102] Figure 33 shows the blood drug concentration-time curve of the antibody-drug conjugate. Detailed Implementation

[0103] Through in-depth research, the inventors have provided a single-domain antibody targeting CAIX, and further provided a bispecific antibody and an antibody-drug conjugate comprising the single-domain antibody. The single-domain antibody has high specificity and good affinity for CAIX, the bispecific antibody has strong endocytosis, and the antibody-drug conjugate has significant cell-killing activity and tumor-suppressing ability. Based on this, the present invention was completed.

[0104] The term "antigen" is a predetermined antigen that an antibody can selectively bind to. Target antigens can be peptides, proteins, nucleic acids, cells, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments described herein, the target antigen is a CAIX protein, including full-length and truncated CAIX proteins.

[0105] Immunoglobulins (Ig) are a class of globulins synthesized and secreted by plasma cells. They possess antibody activity or have a chemical structure similar to antibodies, and are mainly found in blood, tissue fluid, and exocrine fluids. They are core molecules in the humoral immune system. The classic immunoglobulin (Ig) structure is a symmetrical tetrapeptide chain composed of two identical heavy chains (H chains) and two identical light chains (L chains) linked by disulfide bonds, forming a "Y" shape. It contains functional regions such as a variable region, a constant region, and a hinge region; some types also form multimeric structures. Based on the amino acid composition and antigenicity differences of the heavy chain's constant region, immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE. Each heavy chain is divided into a variable region (VH) and a constant region (CH). The light chains are divided into κ-type and λ-type, and each light chain is also divided into a variable region (VL) and a constant region (CL). The amino acid sequence of the variable region is highly variable, with the degree of variability concentrated in the three complementarity-determining regions (CDR1, CDR2, and CDR3). The spacer sequences between the CDR sequences are called frame regions (FRs), and their sequences are relatively conserved. The complementarity-determining regions (CDR1, CDR2, and CDR3) are the sites that directly bind to the antigenic epitope.

[0106] In this article, the term "HCDR" refers to the CDR derived from the heavy chain of classical immunoglobulins (Ig), while "LCDR" refers to the CDR on the light chain. In classical immunoglobulins (Ig), antigen recognition and binding activity are typically determined by both HCDR and LCDR.

[0107] In this paper, the term "single-domain antibody (VHH)" refers to the variable region of the heavy chain of a cloned antibody (Harmsen MM, et al. Appl Microbiol Biotechnol. 2007 Nov; 77(1):13-22). Unlike the classic immunoglobulin (Ig) mentioned above, VHH does not contain a light chain, and its general structure or sequence can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. VHH can have the activity of specifically recognizing and binding to specific antigens without the need for a light chain. VHH is usually obtained from alpaca immune serum by first obtaining antibodies that are naturally missing the light chain and the constant region 1 (CH1) of the heavy chain, and then cloning the variable region of the antibody heavy chain to construct VHH consisting of only a single heavy chain variable region.

[0108] In this document, the terms “single-domain antibody”, “heavy chain single-domain antibody”, “VHH domain”, “VHH”, “VHH antibody fragment”, “VHH antibody”, and “nanobody” (“Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.

[0109] The term "VH" stands for heavy chain variable region, and "VL" stands for light chain variable region.

[0110] The term "full-length antibody" usually refers to the complete form of classical immunoglobulin (Ig).

[0111] The term "antigen-binding fragment" refers to a fragment derived from a full-length antibody that possesses the same binding activity and specificity as the full-length antibody, including but not limited to Fab and scFv. A Fab fragment consists of the variable region domains (VH) of both the L and H chains and the first constant domain (CH1) of one heavy chain. For example, the classic Fab fragment can be obtained by proteolytic hydrolysis of immunoglobulin (Ig).

[0112] The term "single-chain antibody (scFv)" generally refers to an antibody fragment composed of a heavy chain variable region and a light chain variable region linked together (via a linker peptide). Sequentially, the C-terminus of the heavy chain variable region (via a linker peptide) may be linked to the N-terminus of the light chain variable region, or vice versa. The linker peptide is preferably selected from a flexible polypeptide chain composed of alanine and / or serine and / or glycine, and its length can be 3–40 amino acids.

[0113] The term "IMGT numbering system" is an integrated information system specifically for immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complexes (MHC) in humans and other vertebrates. (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). The antibody light and heavy chain genes were analyzed using IMGT (http: / / www.imgt.org / IMGT_vquest) to determine the framework regions (FRs) and complementarity determining regions (CDRs) of the variable domain. The “position” of CDRs within the structure of the immunoglobulin variable domain is conserved across species and is located in structures called loops. Therefore, CDRs and framework residues are easily identified using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into the acceptor framework, typically derived from human antibodies. Unless otherwise stated, in this specification, claims, and drawings, anti-CAIX single-domain antibodies or anti-CD70 antibodies are numbered according to the IMGT numbering method to determine the CDR and FR regions.

[0114] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding module to bind to a specific antigenic determinant can be detected by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and immunofluorescence.

[0115] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from a non-human immunoglobulin, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused to a constant domain, or only a complementarity-determining region (CDR) transplanted into an appropriate scaffold region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble a human immunoglobulin. Some forms of humanized antibodies retain the entire CDR sequence (e.g., humanized single-domain antibodies containing all three CDRs derived from alpacas). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0116] The terms "bispecific antibody," "bispecific antibody," or "bispecific antibody (BsAb)" refer to antibodies with two binding sites that target two different antigens or two different epitopes on the same antigen. The two antigens in a bispecific antibody can be located on the same cell surface or on different cell surfaces. Because bispecific antibodies have two binding sites targeting different antigens or simultaneously recognizing two different epitopes of an antigen, their functional pathways are highly diverse.

[0117] "Immunoconjugates" or "immunocouplers" generally refer to molecules formed by conjugating one or more immunoglobulin-related molecules or fragments thereof (such as antibodies or fragments thereof) with one or more other molecules. These other molecules can be protein-based, such as polypeptides or proteins, or non-protein-based, such as chemical toxins, therapeutic agents, diagnostic agents, radioactive elements, probes, or signaling molecules.

[0118] In a broad sense, "antibody-drug conjugates (ADCs)" refer to molecules formed by covalently linking a therapeutically active drug (payload) to an antibody or its antigen-binding fragment via a linker, enabling drug delivery to the target site to exert its pharmacological function and achieve targeted therapy. Therapeutically active drugs include, but are not limited to, cytotoxic drugs, molecular gels, PROTAC molecules, immune agonists, and radioisotopes. In a narrower sense, ADCs refer to a three-component system consisting of an antibody, a linker, and a cytotoxic drug, including monoclonal antibody ADCs and bispecific / multispecific ADCs, which utilize antibodies to specifically kill cancer cells while reducing toxicity to normal tissues. An ideal ADC should have the following three core components: a highly selective anti-tumor-associated antigen antibody (whose target expression is limited or absent in normal (healthy) cells); a potent cytotoxic payload (usually a small molecule drug with high systemic toxicity) that can induce cell death after endocytosis in target cells; and a linker that is stable in circulation but effectively releases the drug within the target cells.

[0119] "Cytotoxic drugs," or "toxins" or "charges," refer to drugs that can disrupt or inhibit cell growth and proliferation. These drugs are commonly used in cancer treatment because they can kill rapidly dividing cancer cells. The mechanisms of action of cytotoxic drugs are diverse, including but not limited to DNA damage (directly damaging the DNA of cancer cells, preventing its replication and transcription, leading to cell death), microtubule disruption (affecting microtubule formation during cell division, inhibiting the cell cycle), topoisomerase inhibition (inhibiting DNA topoisomerase, the enzyme responsible for uncoiling the supercoiled structure of DNA; inhibiting its activity leads to DNA damage), and nucleic acid synthesis inhibition (blocking the synthesis of DNA or RNA, preventing cell replication). Therefore, cytotoxic drugs include, but are not limited to, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof. Examples include aurestatins (e.g., MMAE and MMAF), camptothecin and its derivatives, taxanes, benzodiazepines, chlortetracycline, methimazole, methimazole A-chain, cobustatin, docamycin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonadoxin (PE)A, PE40, abrin, abrin A-chain, saccharin A-chain, α-diatomaceous, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxamycin, curicin, croton toxin, chachiin, and derivatives of the aforementioned toxins.

[0120] The terms "linker" or "connector" are used interchangeably and refer to the covalently linked unit between an antibody and a drug. The choice of linker has a significant impact on the stability of the antibody-drug conjugate (ADC) and the release of toxins, further affecting efficacy and safety. An ideal linker neither causes ADC aggregation nor hinders ADC stability under physiological conditions, and releases toxins upon reaching tumor cells. Linkers include cleavable and non-cleavable linkers. For non-cleavable linkers, after endocytosis into the lysosomes of the cell, the antibody releases the drug through proteolysis. The released drug often retains the linker structure or even amino acids from the antibody, resulting in poor permeability and hindering the bystander effect. Cleavable linkers include enzyme-cleavable linkers (e.g., peptide or amide bonds that can be degraded by cathepsins, phosphatases, (aryl)sulfatases, β-galactosidases, β-glucosidases, and nitroreductases), acid-cleavable linkers (e.g., hydrazone bonds hydrolyzed under acidic conditions), reducing-condition-cleavable linkers (disulfide bonds, etc.), and exogenously stimuli-cleavable linkers (infrared-sensitive, ultraviolet-sensitive, etc.). Cleavable linkers for cathepsins include, but are not limited to, peptides consisting of 2–10 amino acid residues (e.g., Val-Ala, Val-Cit, Ala-Phe, Ala-Ala-Ala, Gly-Gly-Phe-Gly, etc.). Linker arms possess both spacer and linking functions; they are a collection of chemical structural fragments with different functions. Typically, before being linked to an antibody, the linker arm possesses an active group that can react with lysine or cysteine ​​residues (amino or thiol groups) of the antibody, thereby coupling it to the antibody. Active groups that can react with thiol or amino groups can be selected from maleimides, pyridyl disulfides, haloacyl groups, haloamides, haloesters, iodoacetamides, acyl compounds, methanesulfonylpyrimidines, methanesulfonylpyridines, benzyl halides, polymethylene sulfide thiosulfonates, etc. The spacer group of the linker arm is a key structure connecting the cleavable unit to the load, and can affect drug release kinetics, hydrophobicity, and overall stability. Spacers include self-degradable spacers and hydrophilic modulating spacers. Self-degradable spacers, such as p-aminobenzyloxycarbonyl (PAB), spontaneously decompose chemically after the cleavable unit is cleaved, eliminating steric hindrance of the drug. Hydrophilic modulating spacers, such as PEG, can increase the hydrophilicity of the linker to balance the hydrophobicity of the load and reduce the risk of aggregation.

[0121] The term "DAR" refers to the ratio of the conjugated drug to the antibody. DAR can be an integer or a decimal between 1 and 20, 2 and 10, 3 and 9, or 2 and 8. DAR can also represent the average DAR value in the ADC product (mixture). DAR values ​​can be detected using methods such as HIC-HPLC (hydrochloric spectroscopy), LC-MS (mass spectrometry), RP-HPLC (reversed-phase chromatography), and UV (ultraviolet spectrophotometry). The average DAR value obtained by different detection methods may vary.

[0122] The terms "antibody endocytosis," "antibody endocytosis capacity," or "antibody endocytosis activity" refer to the ability of an antibody to enter the cell through endocytosis after it has specifically bound to an antigen on the cell surface.

[0123] The term "bystander killing effect" refers to the antitumor activity of tumor cells surrounding the target antigen tumor cell, regardless of the target antigen expression status of these surrounding tumor cells.

[0124] The term "blocking activity of an antibody" generally refers to the ability of an antibody to prevent or reduce the normal interaction between the antigen and its receptor or ligand after binding to a specific antigen. This blocking effect can be used for a variety of biological and medical purposes, such as (1) neutralization: In viral infection, antibodies can bind to specific proteins on the surface of the virus, preventing the virus from binding to host cell receptors, thereby neutralizing the virus's infectivity; (2) signal transduction blocking: In the process of cell signal transduction, antibodies can bind to cell surface receptors or specific signaling molecules, blocking signal transmission and affecting cell behavior, such as proliferation and differentiation; (3) immune regulation: Antibodies can be used to regulate the immune system, for example, by blocking specific immune cell surface molecules, changing the intensity or direction of the immune response.

[0125] "Sequence identity" indicates the percentage of identical amino acids between two polypeptide sequences. Methods for evaluating the degree of sequence identity between amino acid sequences are known to those skilled in the art. Amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program in the NCBI database can be used to determine sequence identity.

[0126] The term "composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain any other ingredients that would be unacceptably toxic to a subject receiving the pharmaceutical composition.

[0127] The term "pharmaceuticalally acceptable carrier" refers to a component in a pharmaceutical composition that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0128] The term "treatment / prevention" (and its grammatical variations) refers to an attempt to alter the natural course of a disease in an individual, and can be a clinical intervention implemented for prevention or during the course of clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of symptoms.

[0129] Single-domain antibodies

[0130] A first aspect of this invention provides an anti-CAIX single-domain antibody. The anti-CAIX single-domain antibody comprises CDR1 to CDR3 as shown below:

[0131] The amino acid sequence of CDR1 as shown in SEQ ID NO.1 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with it; the amino acid sequence of CDR2 as shown in SEQ ID NO.2 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with it; or the amino acid sequence of CDR3 as shown in SEQ ID NO.3; or

[0132] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more or 100% sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0133] In some embodiments, the anti-CAIX single-domain antibody provided by the present invention is an alpaca-derived antibody. In some embodiments, the anti-CAIX single-domain antibody provided by the present invention may be a humanized antibody, wherein the humanized antibody retains the functional characteristics of at least one antibody. To further reduce the potential immunogenicity risk of single-domain antibodies during disease treatment, the specific amino acid sequence in the frame region FR of the alpaca-derived antibody can be replaced as much as possible with the amino acid sequence at the corresponding position of the human antibody. The antibody humanization process in this application refers to the chapter on camel-derived single-domain antibody humanization in the book "Single-Domain Antibodies" by Greg Hussack and Kevin A. Henry. Immunogenicity analysis software (http: / / tools.iedb.org / deimmunization / ) and homology modeling software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) were used to predict the candidate sites that need to be replaced for the CAIX single-domain antibody. Referring to the VHH humanization universal framework transplantation method established by Ce′cile Vincke et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath KJ Biol Chem. 2009; 284(5): 3273–3284), the amino acids of the candidate sites were replaced with humanized amino acids.

[0134] The anti-CAIX single-domain antibody of the present invention comprises FR1 to FR4 as shown below: FR1 with amino acid sequences as shown in SEQ ID NO. 22 to 25, FR2 with amino acid sequences as shown in SEQ ID NO. 26 to 37, FR3 with amino acid sequences as shown in SEQ ID NO. 38 to 47, and FR4 with amino acid sequences as shown in SEQ ID NO. 48 to 49.

[0135] In some embodiments of the present invention, the anti-CAIX single-domain antibody comprises FR1 to FR4 as shown below:

[0136] The amino acid sequences are as shown in SEQ ID NO. 22 for FR1, SEQ ID NO. 26 for FR2, SEQ ID NO. 38 for FR3, and SEQ ID NO. 49 for FR4; or

[0137] The amino acid sequences are as shown in SEQ ID NO. 23 for FR1, SEQ ID NO. 31 for FR2, SEQ ID NO. 41 for FR3, and SEQ ID NO. 49 for FR4; or

[0138] The amino acid sequences are as shown in SEQ ID NO. 22 for FR1, SEQ ID NO. 32 for FR2, SEQ ID NO. 42 for FR3, and SEQ ID NO. 48 for FR4; or

[0139] The amino acid sequences are as shown in SEQ ID NO.22 for FR1, SEQ ID NO.35 for FR2, SEQ ID NO.44 for FR3, and SEQ ID NO.49 for FR4.

[0140] The amino acid sequences are as shown in SEQ ID NO.25 for FR1, SEQ ID NO.35 for FR2, SEQ ID NO.44 for FR3, and SEQ ID NO.49 for FR4.

[0141] In some embodiments of the present invention, the amino acid sequence of the anti-CAIX single-domain antibody includes: a) an amino acid sequence as shown in any one of SEQ ID NO. 7 to 21; or, b) an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with any one of SEQ ID NO. 7 to 21 and having the function of the amino acid sequence defined in a); specifically, the amino acid sequence in b) specifically refers to: an amino acid sequence as shown in any one of SEQ ID NO. 7 to 21 obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or obtained by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having amino acids as shown in SEQ ID NO. 7 to 21. The polypeptide fragments shown in one of No. 7 to 21 are functional polypeptide fragments, for example, the ability to specifically bind to CAIX.

[0142] Fusion protein

[0143] The present invention provides a fusion protein comprising a first domain, which is an anti-CAIX single-domain antibody as described above; and a second domain, which has the effect of prolonging the in vivo half-life and / or binding to effector cells.

[0144] In some embodiments, the second domain includes one or more of a serum albumin fragment, a polyethylene glycol fragment, and a nanobody that binds HSA; and / or, the second domain includes an immunoglobulin Fc region; and / or, the second domain includes a molecule that has affinity for CD3 present on T cells and / or is capable of binding to CD3 present on T cells.

[0145] In some embodiments, the immunoglobulin Fc region is the human immunoglobulin Fc region, the immunoglobulin is selected from one or more combinations of IgG, IgGA1, IgGA2, IgD, IgE, and IgM, and the IgG is selected from one or more combinations of IgG1, IgG2, IgG3, or IgG4 subtypes.

[0146] Bispecific antibodies

[0147] This invention provides a novel bispecific antibody (biantibody) structure, wherein one arm is anti-CAIX VHH and the other arm is anti-CD70 Fab. The biantibody has the following structural features:

[0148] The first heavy chain, consisting of the anti-CAIX VHH, is linked to the Fc region of human IgG1 via the hinge region. The second heavy chain, consisting of the anti-CD70 monoclonal antibody Vorsetumumab, is linked to the constant region of the human IgG1 heavy chain (CH1 + hinge region + Fc). The light chain is the light chain of Vorsetumumab. The two Fc chains form a heterodimer through a Knob-in-Hole. Alternatively, the C-terminus of the first heavy chain, the anti-CAIX VHH, is linked to the human IgG1 Fc via the hinge region. The left arm heavy chain (second heavy chain), consisting of the C-terminus of the anti-CD70 monoclonal antibody VH, is linked to the constant region of the human IgG1 heavy chain (CH1 + hinge region + Fc). The left light chain is the light chain of Vorsetumumab. The left and right arms form a heterodimer through a Knob-in-Hole. The bispecific antibody exhibits lower binding activity to CAIX and CD70 than that of anti-CAIX parent monoclonal antibodies (e.g., 13E6-Fc) and anti-CD70 monoclonal antibodies (e.g., Vorsetumumab). This reduces the binding of the bispecific antibody to normal tissues, which helps to reduce systemic toxicity. At the same time, the bispecific antibody can bind with high affinity to tumor cells co-expressing CAIX and CD70, improving tumor targeting and enhancing therapeutic efficacy.

[0149] In some specific embodiments of the present invention, the CAIX / CD70 bispecific antibody includes an anti-CAIX single-domain antibody comprising:

[0150] The amino acid sequence is as shown in SEQ ID NO.1 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.2 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.3 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3; or

[0151] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more or 100% sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0152] In some specific embodiments of the present invention, the CAIX / CD70 bispecific antibody contains an anti-CD70 antibody or its antigen-binding fragment comprising HCDR1 as shown in SEQ ID NO. 67, HCDR2 as shown in SEQ ID NO. 68, HCDR3 as shown in SEQ ID NO. 69, LCDR1 as shown in SEQ ID NO. 70, LCDR2 as shown in SEQ ID NO. 71, and LCDR3 as shown in SEQ ID NO. 72.

[0153] In some specific embodiments, the bispecific antibody comprises:

[0154] The first heavy chain shown in SEQ ID No. 86, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0155] The first heavy chain shown in SEQ ID No. 87, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0156] The first heavy chain shown in SEQ ID No. 88, the second heavy chain shown in SEQ ID No. 90, and the light chain shown in SEQ ID No. 66.

[0157] Isolated polynucleotides

[0158] This invention also provides an isolated polynucleotide encoding a single-domain antibody provided in the first aspect of this invention, a fusion protein provided in the second aspect of this invention, or a bispecific antibody provided in the third aspect of this invention. The polynucleotide may be RNA, DNA, or cDNA, etc. Methods for providing the isolated polynucleotide should be known to those skilled in the art; for example, it can be prepared by automated DNA synthesis and / or recombinant DNA technology, or it can be isolated from a suitable natural source. In one specific embodiment of this invention, the nucleic acid sequence of the isolated polynucleotide is shown in one of SEQ ID Nos. 94 to 113.

[0159] expression carrier

[0160] This invention also provides an expression vector containing the isolated polynucleotides described above. The construction methods of the expression vector should be known to those skilled in the art. For example, the expression vector can be constructed using in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. More specifically, it can be constructed by inserting the isolated polynucleotides into the multiple cloning site of the expression vector. The expression vector in this invention generally refers to various commercially available expression vectors well-known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The vector may also include one or more regulatory sequences operatively linked to the polynucleotide sequence, and the regulatory sequences may include suitable promoter sequences. The promoter sequence is typically operatively linked to the coding sequence of the amino acid sequence to be expressed. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequences may also include suitable transcription terminator sequences, sequences recognized by the host cell to terminate transcription. The terminator sequence is attached to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in a selected host cell can be used in this invention.

[0161] Generally, a suitable vector may contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers. For example, these promoters may include, but are not limited to, the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the Pichia pastoris methanol oxidase promoter, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes may be used to provide phenotypic traits for selecting host cells for transformation; for example, they may include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for *E. coli*. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. If an enhancer sequence is inserted into the vector, transcription will be enhanced. An enhancer is a cis-acting factor of DNA, typically about 10 to 300 base pairs, that acts on the promoter to enhance gene transcription (Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press).

[0162] Expression system

[0163] This invention also provides an antibody expression system, wherein the expression system contains the aforementioned expression vector or a genome in which exogenous polynucleotides are integrated. The expression system is a cell. Any cell suitable for expression using the expression vector can serve as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to bacterial cells of *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*; fungal cells such as yeast, filamentous fungi, and plant cells; insect cells of *Drosophila S2* or *Sf9*; and animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells. Methods for constructing the expression system should be known to those skilled in the art.

[0164] Antibody-drug conjugates

[0165] The present invention also provides an antibody-drug conjugate. The antibody-drug conjugate has the structure shown in formula (I) or a pharmaceutically acceptable salt thereof:

[0166] Ab-(LD) n , formula (I);

[0167] Where Ab is an anti-CAIX single-domain antibody or a CAIX / CD70 bispecific antibody; L is a linker arm; D is a cytotoxic drug; and n is a number between 1 and 20, representing the DAR value, which can be an integer or a decimal.

[0168] In some embodiments, the cytotoxic drug D is selected from one or more of the following: maytansine derivative DM1, maytansine derivative DM4, monomethylolpropionate E (MMAE), monomethylolpropionate F (MMAF), duocarmycin, pyrrolobenzodiazepine (PBD), camptothecin and its derivatives [including but not limited to SN38, eczetine (Exatecan, abbreviated as Exd), Dxd], tubulysins, amanitin, PNU-159682, and calicheamicins.

[0169] In some embodiments, the linker arm L is selected from non-cleavable linkers or cleavable linkers. The cleavable linker arm, depending on the cleavage mechanism, includes enzyme-mediated (e.g., cathepsins, phosphatases, (aryl)sulfatases, β-galactosidases, β-glucosidases, and nitroreductases) cleavable linkers, acid-cleavable linkers, linkers cleavable under reducing conditions (disulfides, etc., where disulfide bonds break under reducing conditions), and linkers cleavable by exogenous stimuli (infrared sensitive, ultraviolet sensitive, etc.). In a specific embodiment of this application, the linker arm is a cleavable linker arm. The antibody conjugate is internalized into tumor cells, and the cleavage of the linker arm allows the release of a therapeutically active drug, such as Exatecan, to kill tumor cells. Furthermore, it can exert a bystander effect, killing surrounding heterogeneous tumor cells.

[0170] In some embodiments, the linker arm L is carbon-based, amino-based, amide-based, acyl-based, or -(PEG). m -、-(CH2) m - Containing heteroatoms -(CH2) m-, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide (Mal or MA), maleimide caproyl (MC), maleimide propoyl (MP), VA (Val-Ala), VC (Val-Cit), VK (Vla-Lys), AF (-Ala-Phe-), GGFG (Gly-Gly-Phe-Gly), p-aminobenzyloxycarbonyl (PAB), N-succinimide 4-(N-maleimide methyl)-cyclohexane-1-carboxylic acid ester (SMCC), N-succinimide 4-(2-pyridinylthio)valerate (SPP), polyethylene glycol (PEG), pyrimidine, pyridine (py), methanesulfonylpyrimidine, methanesulfonylpyridine, and one or more combinations thereof, wherein each m is independently selected from an integer from 1 to 20.

[0171] In some implementations, the connecting arm L includes one or more combinations of the following structures:

[0172] Each q is independently selected from integers from 1 to 20, for example, integers from 1 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0173] X is selected from -NH-, -O-, and -S-;

[0174] Su are each independently selected from pentose, penturonic acid, hexose, and hexuronic acid;

[0175] R c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups.

[0176] In a specific embodiment of this application, one end of the linker arm is linked to a cysteine ​​residue (thiol group) of the antibody, and the other end is linked to a cytotoxic drug.

[0177] In some embodiments, the linker arm L is -MC-VC-PAB- and the cytotoxic drug D is MMAE; or the linker arm L is -MC-GGFG- and the cytotoxic drug D is Dxd; or the linker arm L is -pym-PEG8-VA-PAB- and the cytotoxic drug D is Exatecan.

[0178] In some embodiments, the antibody-drug conjugates of this application are selected from:

[0179] Where Ab represents anti-CAIX single-domain antibody or CAIX / CD70 bispecific antibody; n represents the average DAR value, for example, n is an integer or decimal between 1 and 20.

[0180] In some embodiments, Ab is an anti-CAIX single-domain antibody comprising CDR1 with an amino acid sequence as shown in SEQ ID NO.1 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity, CDR2 with an amino acid sequence as shown in SEQ ID NO.2 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity; or

[0181] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0182] In some embodiments, the Ab is a CAIX / CD70 bispecific antibody, wherein the anti-CAIX single-domain antibody comprises: CDR1 with an amino acid sequence as shown in SEQ ID NO.1 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity therewith; CDR2 with an amino acid sequence as shown in SEQ ID NO.2 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity therewith; and CDR3 with an amino acid sequence as shown in SEQ ID NO.3 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity therewith; or

[0183] The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

[0184] The CD70-targeting domain is an anti-CD70 antibody or its antigen-binding fragment, which contains HCDR1 as shown in SEQ ID NO. 67, HCDR2 as shown in SEQ ID NO. 68, HCDR3 as shown in SEQ ID NO. 69, LCDR1 as shown in SEQ ID NO. 70, LCDR2 as shown in SEQ ID NO. 71, and LCDR3 as shown in SEQ ID NO. 72.

[0185] In some embodiments, the Ab is a CAIX / CD70 bispecific antibody, which comprises:

[0186] The first heavy chain shown in SEQ ID No. 86, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0187] The first heavy chain shown in SEQ ID No. 87, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or

[0188] The first heavy chain shown in SEQ ID No. 88, the second heavy chain shown in SEQ ID No. 90, and the light chain shown in SEQ ID No. 66.

[0189] Composition

[0190] The present invention also provides a composition comprising the above-described antibody-drug conjugate or a pharmaceutically acceptable salt thereof. The molar ratio (DAR) of D to Ab in the composition is a number between 1 and 20, preferably an integer or decimal between 1 and 10. In some embodiments, the content of the conjugate in the composition is greater than 50%.

[0191] In some embodiments, the composition may further include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include, but are not limited to, sterile water, buffer solutions, isotonic saline solutions, sugars, nonionic surfactants, etc.

[0192] use

[0193] The present invention provides the use of the single-domain antibody described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the bispecific antibody provided in the third aspect of the present invention, the antibody-drug conjugate described in the fourth aspect of the present invention, or the composition provided in the fifth aspect of the present invention in the preparation of a medicament for the diagnosis, treatment, or prevention of CAIX-positive or CD70-positive tumors.

[0194] The "therapeutic effective amount" of the single-domain antibodies, fusion proteins, bispecific antibodies, antibody-drug conjugates, and pharmaceutical compositions provided by this invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability caused by disease-related suffering. For example, for the treatment of CAIX-related tumors, the "therapeutic effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80% relative to untreated subjects. The ability to inhibit tumor growth can be evaluated in animal model systems that predict the efficacy of treatment for human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays known to those skilled in the art. Therapeutic effective amounts of single-domain antibodies, fusion proteins, and pharmaceutical compositions generally reduce tumor size or otherwise alleviate symptoms in subjects. This invention can be used for the diagnosis, treatment, or prevention of CAIX-positive tumors, including but not limited to renal cell carcinoma, breast cancer, lung cancer, liver cancer, and colorectal cancer.

[0195] Treatment

[0196] The sixth aspect of the present invention also provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of any one or more of the following substances: a single-domain antibody as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention, or a bispecific antibody as described in the third aspect of the present invention, or an antibody-drug conjugate as described in the fourth aspect of the present invention, or a composition as described in the fifth aspect of the present invention.

[0197] "Subjects" include, but are not limited to, animals, preferably mammals; said mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep or other non-human mammals; non-mammals include, for example, non-mammal vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammal invertebrates.

[0198] In some embodiments of the present invention, the tumor is selected from renal cell carcinoma, breast cancer, lung cancer, liver cancer, colorectal cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, malignant lymphoma, pancreatic cancer, glioblastoma, etc.

[0199] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, achieving complete or partial reversal of the condition, curing the condition, or a combination of the above. For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0200] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0201] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0202] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0203] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; the series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATIN STRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0204] The abbreviations used in the examples have the following specific meanings:

[0205] BSA: Bovine Serum Albumin;

[0206] PBS: Phosphate-Buffered Saline;

[0207] PBST: Phosphate-Buffered Saline with Tween-20;

[0208] TMB: 3,3',5,5'-Tetramethylbenzidine;

[0209] EGFP: Enhanced Green Fluorescent Protein;

[0210] HRP: Horseradish Peroxidase;

[0211] Goat Anti-Human IgG, Monkey ads-HRP: Goat anti-human IgG secondary antibody, adsorbed onto monkey serum and labeled with HRP;

[0212] Anti-M13 Antibody (HRP): HRP-labeled antibody against M13 phage;

[0213] DAR: Dye-to-Antibody Ratio, which is the ratio of dye molecules to antibody molecules in fluorescently labeled antibodies, or the ratio of toxin molecules to antibody molecules in antibodies conjugated with toxins.

[0214] Goat Anti-Mouse IgG H&L ( 650)preadsorbed: Goat anti-mouse IgG H&L ( 650) pre-adsorbed secondary antibody;

[0215] Goat Anti-Human IgG Fc Cross-Adsorbed Secondary Antibody( 650): Goat anti-human IgG Fc ( 650) fluorescent secondary antibody;

[0216] PNPA: 4-Nitrophenylacetic acid ester;

[0217] HT-29: Human colon cancer cells;

[0218] HT-29-CAIX: Human colon cancer cells that overexpress CAIX;

[0219] H1975: Human lung adenocarcinoma cells;

[0220] OSRC-2: Human kidney cancer cells;

[0221] OSRC-2-CAIX: Renal cancer cells in humans that overexpress CAIX;

[0222] 786-O: Human renal cell adenocarcinoma cells;

[0223] 786-O-CAIX: Human renal cell adenocarcinoma cells overexpressing CAIX (CAIX / CD70 high expression);

[0224] CHOK1Q-CAIX: Single-positive CHO cells overexpressing CAIX;

[0225] CHOK1-CD70: Single-positive CHO cells overexpressing CD70.

[0226] Example 1: Construction of Anti-CAIX Single-Domain Antibody Library

[0227] The CAIX sequence (137-390, CA sequence, SEQ ID NO:51) was emulsified with 0.5 mg of CAIX(137-390)-llamaFc (SEQ ID NO:52), a fusion protein of alpaca immunoglobulin G-Fc, and 0.5 mL of Freund's adjuvant (Sigma). Healthy alpacas (Vicugna pacos) were immunized with this mixture, and a second immunization was performed 21 days later, for a total of three immunizations. This stimulated B cells to express antigen-specific single-domain antibodies. Blood samples were collected on day 49 for serum analysis. The antibody titer in the alpaca serum was determined using ELISA. 30 mL of alpaca blood was collected using vacuum blood collection tubes. Lymphocytes were separated using lymphocyte separation fluid (Tianjin Haoyang Huake Biotechnology Co., Ltd.), and total RNA was extracted using the Trizol method. 5 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen) according to the manufacturer's instructions. VHH was then amplified using nested PCR, and the final round of PCR carried VHH onto the vector homologous recombination arm. The target VHH nucleic acid fragment was recovered and recombined into the phage display vector pcomb3xss (Addgene plasmid #63890; RRID: Addgene_63890) using a homologous recombinase (Suzhou Nearshore Protein Technology Co., Ltd.). The homologous recombination product was transformed into electroporation competent cells XL1-Blue (Nanjing Heming Yinggu Biotechnology Co., Ltd.) to construct an Anti-CAIX single-domain antibody library. The library was plated using serial dilutions, and the library volume was determined to be greater than 10^6 cells / mL. 7 Meanwhile, more than 24 clones were randomly selected for Sanger sequencing, and the results showed that the insertion rate of the constructed library was 100% and the sequence diversity of the library was 100%.

[0228] Example 2: Screening and Identification of Anti-CAIX Single-Domain Antibodies

[0229] 2.1 Screening of Anti-CAIX Single-Domain Antibodies

[0230] The constructed Anti-CAIX single-domain antibody library was packaged using helper phage M13KO7 (NEB) to obtain a recombinant phage display library. The fusion protein CAIX(137-390)-TS-His (SEQ ID NO. 53), composed of the CAIX(137-390) sequence and a Twin-Strep-His tag, was used as the coating protein. The protein was diluted to 5 μg / mL with 100 mM NaHCO3 pH 8.2 coating buffer and 100 μL / well was added to coat microplates (Beaverbio). The plates were incubated overnight at 4°C. The next day, 200 μL of 3% BSA was added and the plates were blocked at 37°C for 2 h. Approximately 1 × 10⁻⁶ ppm of the solution was then added. 11 The recombinant phage display library was incubated at 37°C for 2 hours using PFU / well. Afterwards, it was washed 5 times with PBST (PBS containing 0.05% Tween 20) and 10 times with PBS to remove non-specifically bound phages. The washed ELISA wells were eluted with 100 μL / well of 0.1M Gly-HCl 1 mg / mL BSA (pH 2.2) buffer at 37°C for 8 minutes, and then neutralized with 1M pH 8.0 Tris-HCl. The phage titer of the eluted library was measured, and the phage eluent was amplified. CAIX(137-390)-llamaFc was used as the coating protein, the coating amount was reduced to 200 ng / well, and 3% ovalbumin (OVA) was used for blocking. The incubation time was shortened to 37°C × 1 hour, and the washing conditions were enhanced to 10 washes with PBST and 20 washes with PBS for a second round of affinity enrichment. Based on the above basic ideas and methods for phage panning, two or three rounds of enrichment panning were carried out. At the same time, various panning conditions were introduced, including liquid-phase affinity panning and cell affinity panning by constructing cell lines that overexpress human CAIX.

[0231] 2.2 Phage ELISA for the identification of Anti-CAIX single-domain antibodies

[0232] A total of 1248 single clones were picked from phage titer assay plates washed and eluted under different selection conditions and cultured in 96-well plates. M13KO7 helper phage was used for infection and packaging to obtain the accumulation of recombinant phage in the supernatant. CAIX(137-390)-llamaFc was coated at 100 ng / well and blocked with 3% BSA at 37°C for 2 h, or coated with CAIX(137-390)-TS-His for liquid chromatography detection. 100 μL / well of the single-clone recombinant phage supernatant was incubated in 96-well plates coated with CAIX(137-390)-llamaFc or CAIX(137-390)-TS-His (wells containing only M13KO7 helper phage served as negative controls) at 37°C for 1 h. After washing five times with PBST, 100 μL of 0.1 μg / mL Anti-M13 Antibody (HRP) and Mouse Monoclonal (SinoBiological) were added to each well, and incubated at 37°C for 1 hour. After washing five times with PBST, TMB chromogenic working solution (Huzhou Yingchuang Biotechnology Co., Ltd.) was added, and after incubation at 37°C for 5 minutes, the reaction was terminated by adding 1M sulfuric acid, and the OD450nm value was recorded. The M13KO7 negative control wells showed weak or almost no color development (OD450nm value below 0.3), while the CAIX (137-390) positive wells showed significant color development or a significant difference compared to the negative wells (OD450nm value above 2.5). Positive clones were selected for Sanger sequencing, and repetitive sequences were removed.

[0233] After multiple rounds of screening, two anti-CAIX single-domain antibody clones with strong or relatively strong positive binding to CAIX (137-390) were selected: 3H4 and 13E6. Their full-length sequences are shown in Table 1, where the underlined regions indicate the CDR regions.

[0234] Table 1

[0235] Example 3 Expression and purification of Anti-CAIX-Fc fusion protein

[0236] To facilitate the characterization of CAIX VHH's affinity, specificity, and other properties, a detection tag (e.g., Fc, His, Twin-Strep-tag, Myc, GST, Flag, or HA) can be bound to anti-CAIX VHH. In this invention, Fc is selected as the detection tag. CAIX VHH is fused with Fc to construct a dimer similar to a traditional antibody. Therefore, the choice of tag protein and Fc sequence should not be a limitation of this invention. In this embodiment, Anti-CAIX-3H4 / Anti-CAIX-13E6 are respectively linked to the human IgG1 Fc region (SEQ ID NO. 75) to construct the Anti-CAIX-3H4-Fc and Anti-CAIX-13E6-Fc fusion proteins, wherein the Fc region includes a hinge region. The full-length sequences of Anti-CAIX-3H4-Fc, Anti-CAIX-13E6-Fc, reference antibody G119 Analog (Chen Xu, et al. PLoS One. 2010 Mar 10; 5(3)), and reference antibody Hu-H-YE-32-D5 analog (derived from patent CN 116217728 A) are shown in Table 2.

[0237] Table 2

[0238] The above sequence was synthesized and inserted into the pcDNA3.1 expression vector. The expression vector and transfection reagent PEI (Yisheng Biotechnology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a ratio of 1:3 and cultured at 37°C in a 5% CO2 shaker incubator for 7 days. The expressed protein or antibody was recovered by centrifugation and purified by Protein A affinity chromatography column (Borglon Biotechnology Co., Ltd.). The target antibody was obtained by purity detection and quantitative analysis.

[0239] Example 4: Binding ability of Anti-CAIX-Fc fusion protein to recombinant human CAIX protein

[0240] CAIX(137-390)-TS-His was coated at 100 ng / well and incubated overnight at 4°C. The plates were then blocked with 3% BSA at 37°C for 2 hours. The Anti-CAIX-3H4-Fc / Anti-CAIX-13E6-Fc fusion protein and positive control antibody were serially diluted with 1% BSA and incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of 0.1 μg / mL Goat Anti-Human IgG, Monkey ads-HRP (Southern Biotech) was added to each well, and the plates were incubated at 37°C for 1 hour. After washing five times with PBST, TMB substrate was added, and the plates were incubated at 37°C. After 5 minutes of incubation and color development, the reaction was stopped by adding 1M sulfuric acid. The OD450nm reading was used to calculate the EC50 of the anti-CAIX antibody against the recombinant human CAIX(137-390) protein. 50 The values ​​and experimental results are shown in Figures 1A-1B, Table 3-1, and Table 3-2.

[0241] Table 3-1

[0242] Table 3-2

[0243] Example 5: Binding ability of Anti-CAIX-Fc fusion protein to CAIX-overexpressing cells

[0244] An expression plasmid containing the full-length human CAIX gene sequence (SEQ ID NO. 50) linked to the EGFP fluorescent protein gene sequence was constructed. This expression vector contains NeoR / KanR and can be used for G418 drug screening. The CAIX (SEQ ID NO: 54) gene sequences of cynomolgus monkeys and the CAIX (SEQ ID NO: 55) gene sequences of mice were combined with the EGFP fluorescent protein gene sequence to construct corresponding overexpression vectors. These expression vectors contain glutamine synthase (GS) and can be used for glutamine synthase inhibitor (MSX) drug screening.

[0245] The expression plasmids containing the human CAIX gene sequence were electroporated into CHOK1Q cells, and the CHOK1Q-CAIX cell line was obtained by G418 pressure selection and indefinite dilution. Transiently overexpressing cynomolgus macaque CAIX cells (Expi293F-cynoCAIX) or overexpressing mouse CAIX cells (Expi293F-mCAIX) were obtained by transiently transfecting the expression plasmids containing the cynomolgus macaque or mouse CAIX gene sequences into Expi293F cells. 5 × 10⁶ cells were seeded per well in 96-well plates. 5One overexpressing cell line was centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and serially diluted test antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL PBS, and 100 μL of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody (DyLight) was added. TM Incubate at 650 μL (Invitrogen, diluted 1:300 with 1% BSA) at 4°C for 1 hour. Centrifuge to remove supernatant, and wash three times with 200 μL PBS. Resuspend cells in 100 μL PBS, and detect antibody binding rate and mean fluorescence intensity using flow cytometry (Agilent 2060R). The affinity of Anti-CAIX-Fc fusion protein for human CAIX overexpressing cells CHOK1Q-CAIX is shown in Table 4. Cross-tests of Anti-CAIX-Fc fusion protein (10 μg / mL) for cynomolgus monkey CAIX overexpressing cells CHOK1Q are shown in Table 5-1, and cross-tests of Anti-CAIX-Fc fusion protein (10 μg / mL) for mouse CAIX overexpressing cells Expi293F-cynoCAIX are shown in Table 5-2 (cynomolgus monkey cynoCAIX). Cross-tests of Anti-CAIX-Fc fusion protein (10 μg / mL) for mouse CAIX overexpressing cells Expi293F-mCAIX are shown in Table 6 (mouse CAIX).

[0246] Anti-CAIX-3H4-Fc and Anti-CAIX-13E6-Fc antibodies exhibit strong binding ability to CAIX expressed on the cell surface of humans, cynomolgus monkeys, or mice, comparable to the reference standard.

[0247] Table 4

[0248] Table 5-1

[0249] Table 5-2

[0250] Table 6

[0251] Example 6: Binding of Anti-CAIX-Fc fusion protein to tumor cells

[0252] In a 96-well plate, inoculate 3 × 10⁶ cells per well. 5Tumor cells (HT-29 cells, colo205 cells, OS-RC2 cells) were collected. Centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and 10 μg / mL diluted antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL PBS, and 100 μL of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody (DyLight) was added. TM Incubate at 650 μL (Invitrogen, diluted 1:300 with 1% BSA) at 4°C for 1 hour. Centrifuge to remove supernatant, and wash three times with 200 μL PBS. Resuspend cells in 100 μL PBS, and detect antibody binding rate and mean fluorescence intensity using flow cytometry (Agilent 2060R). The binding of the Anti-CAIX-Fc fusion protein to tumor cells is shown in Table 7. All antibodies bound tumor cells normally.

[0253] Table 7

[0254] Example 7: Detection of the inhibitory effect of the Anti-CAIX-Fc fusion protein on CAIX enzyme activity.

[0255] CAIX catalyzes the conversion of carbon dioxide to bicarbonate, but its actual enzyme activity is difficult to detect. Referring to the method of V Hovanky & KMehta et al. (Journal of Young Investigators, 2014 Vol. 27 Issue 2), 4-nitrophenylacetate was used instead of the reaction substrate. 5 μg of each antibody protein was mixed with 5 μg of CAIX (137-390)-llamaFc protein in a 50 μL system, and incubated in 96-well plates at 37°C for 20 min. Then, 50 μL of buffer containing 2 nM 4-nitrophenylacetate was added to each well of the 96-well plate, resulting in a final reaction concentration of 1 nM 4-nitrophenylacetate. Microplate reader readings were taken at OD400 at room temperature, with readings every 30 seconds for 16 min. The fitted curves are shown in Figure 2. 3H4 and 13E6 showed better inhibitory activity against CAIX than the reference antibody Hu-H-YE-32-D5 analog, while G119 analog showed no significant inhibitory activity.

[0256] Example 8: Detection of the binding ability of the Anti-CAIX-Fc fusion protein to other proteins in the CA family.

[0257] CAXII (SEQ ID NO. 54) of the CAs family is expressed in the kidney, intestine, germinal epithelium, eye, and tumors, while CAXIV (SEQ ID NO. 55) is expressed in the kidney, brain, liver, and skeletal muscle. CAXII and CAXIV are two transmembrane proteins in the CAs family, excluding CAIX. Other family members are mainly located in the cytosol or mitochondria and have low homology with CAIX (less than 40%). Therefore, the cross-binding activity of the Anti-CAIX-Fc fusion protein with CAXII and CAXIV was primarily considered. The CAXII and CAXIV genes were synthesized and constructed into pcDNA3.1, linked to GFP via P2A. Transient overexpression of CAXII and CAXIV was obtained by transiently transfecting Expi293F cells with the expression plasmids containing the CAXII and CAXIV gene sequences.

[0258] In a 96-well plate, inoculate 3 × 10⁶ cells per well. 5 Expi293F cells were transiently overexpressing CAXII and CAXIV. After centrifugation at 1000 rpm for 5 minutes, the supernatant was removed, and 10 μg / mL of diluted test antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL PBS, and 100 μL of Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody (DyLight) was added. TM Cells were incubated at 650 μL (Invitrogen, diluted 1:300 with 1% BSA) at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL PBS. Cells were resuspended in 100 μL PBS, and antibody binding rate and mean fluorescence intensity were detected by flow cytometry (Agilent 2060R). The binding of the Anti-CAIX-Fc fusion protein to CAXII and CAXIV is shown in Table 8. The antibodies did not cross-bind to CAXII or CAXIV, indicating good antibody specificity.

[0259] Table 8

[0260] Example 9: Humanization of Anti-CAIX VHH

[0261] To further reduce the potential immunogenicity risk of single-domain antibodies during disease treatment, specific amino acid sequences in the camel-derived antibody framework are replaced as much as possible with amino acid sequences at positions corresponding to those in the human antibody. In this application, the antibody humanization method adopts the VHH humanization universal framework transplantation method established by Ce′cile Vincke et al. (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath KJ Biol Chem. 2009; 284(5): 3273–3284). In addition, referring to the camel-derived single-domain antibody humanization chapter in the book "Single-Domain Antibodies" by Greg Hussack and Kevin A. Henry, the immunogenicity analysis software (http: / / tools.iedb.org / deimmunization / ) and homology modeling software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) were used to predict the candidate sites that need to be replaced, and the amino acids of the candidate sites were replaced with humanized amino acids. This embodiment only lists a few representative humanized sequences; many more humanization possibilities exist, which can be obtained by those skilled in the art using the methods described in this embodiment or conventional humanization methods. Exemplary antibody sequences before and after humanization are shown in Table 9. "V1, V2, V3, V4" represent different humanized versions and are not case-sensitive. For ease of characterization, the humanized antibody Fc fusion protein was constructed by linking it to the human IgG1 Fc region (SEQ ID NO. 75). Furthermore, the inventors also performed various mutations on the FR region of hu13E6V2 to further study the activity of the FR variants. The sequences of various FR variants of hu13E6V2 are also shown in Table 9.

[0262] Table 9

[0263] The above-mentioned humanized gene sequences were synthesized and transiently transfected into Expi293F cells for expression. The supernatant of the expressed antibody was recovered by centrifugation and purified by Protein A affinity chromatography column (BorgLon Biotech Ltd.). The target humanized antibody was obtained by purity detection and quantitative analysis.

[0264] Following the methods in Examples 4 and 5, affinity was verified by flow cytometry and ELISA. The results are shown in Figure 3 (CHOK1Q-huCAIX cells), Figure 4 (huCAIX(137-390)-TS-His fusion protein), and Figure 5 (CHOK1Q-huCAIX cells). The affinity did not change significantly before and after humanization, and the affinity of the humanized antibodies hu13E6V2-2D1, hu13E6V2-2E4, hu13E6V2-3A5, and hu13E6V2-3B8 derived from hu13E6V2 was slightly better than that of 13E6.

[0265] Example 10: Endocytotic activity of humanized Anti-CAIX VHH

[0266] To ensure homogeneity, alpaca VHH cells specifically binding to human IgG1 Fc were labeled using pHAb Amine Reactive Dye (promega, REF: G9845). The labeled VHH cells were designated pHAb-lgG-VHH-HisK. 25 μL of pHAb-lgG-VHH-HisK was then added to 2 mL of culture medium, aliquoted into 100 μL portions, and 1.5 μg of aCAIX-13E6 humanized antibody was added to each portion. After incubation at 37°C for 1 h, 30,000 CHOK1Q-CAIX cells were resuspended and incubated at 37°C with 5% CO2 for 20 h. The medium was changed to PBS, and the results were photographed (Figure 6). The results showed that the humanized antibody still maintained strong endocytic capacity.

[0267] Example 11 pH bias of Anti-CAIX VHH

[0268] Since high expression of CAIX in tumor tissues reduces extracellular pH and the tumor microenvironment becomes more acidic, the tumor cell binding activity of candidate antibodies at different pH levels was detected by FACS according to the method in Example 6 to evaluate whether the candidate antibodies have pH bias.

[0269] The binding activities of candidate antibodies and HT29 cells under different pH conditions (pH 6.0, pH 7.5) are shown in Figure 7A: the 13E6 antibody binds more strongly (about 5 times) at pH 6.0 than at pH 7.5, showing a certain pH bias. The antibody tends to bind to tumor cells in the slightly acidic environment of the tumor, reducing its binding to normal cells in vivo and reducing peripheral toxicity. Hu-H-YE-32-D5 and Girentuximab, on the other hand, do not show pH bias.

[0270] CA9hu-1 is a monoclonal antibody in Phase I clinical trials (Zatovicova, et al. (2022) Cancer Metab 10,3). The binding activity of CA9hu-1 to CHOK1Q-CAIX cells under different pH conditions (pH 6.0, pH 7.2) is shown in Figure 7B: the binding activity of CA9hu-1 is similar under pH 6.0 and pH 7.2 conditions, without pH bias, and the affinity of CA9hu-1 is also significantly lower than that of the antibody provided in this invention.

[0271] Example 12 Preparation of CAIX / CD70 bispecific antibody

[0272] 12.1 Construction and Expression

[0273] Bispecific antibodies were prepared by combining a single-domain antibody targeting CAIX and an antibody targeting CD70. An exemplary bispecific antibody structure of this invention is shown in Figure 8. Following the relevant sequences shown in Table 10, the DNA sequence corresponding to DR31503 was optimized and synthesized by Jiangsu Huakang Biotechnology Co., Ltd. The heavy chain Knob and Hole, as well as the light chain, were inserted into the pcDNA3.4 vector containing the signal peptide, resulting in expression plasmids pcDNA3.4-DR31503-Knob, pcDNA3.4-DR31503-Hole, and pcDNA3.4-DR31503-LC, consisting of the light chain, the first heavy chain (a heavy chain fused with Anti-CAIX VHH), and the second heavy chain.

[0274] Expi293F cells were passaged and expanded using CD01 medium (Hangzhou Peiding Biotechnology Co., Ltd.) until the cell density reached 2–4 × 10⁻⁶ cells / year. 6 Viable cells / mL, viability ≥95%. Light chain and heavy chain expression plasmids pcDNA3.4-DR31503-Knob, pcDNA3.4-DR31503-Hole, and pcDNA3.4-DR31503-LC were extracted using an endotoxin-free plasmid extraction kit. After sterilization by filtration through a 0.22 μm filter membrane, the plasmids were mixed at a 1:1:1 ratio. The expression plasmids and transfection reagent PEI (Yisheng Biotechnology (Shanghai) Co., Ltd.) were transfected into Expi293F cells (Thermo) at a 1:3 ratio. The cells were cultured at 37°C in a 5% CO2 shaker incubator for 7 days, and the culture supernatant was harvested.

[0275] The construction and expression of DR31506 and DR31507 are the same as those of DR31503.

[0276] Table 10

[0277] 12.2 Purification

[0278] The cell fermentation supernatant was loaded onto an AT Protein A Diamond affinity chromatography column (Borglon Biotechnology Co., Ltd.), with a residence time of 2 min. The equilibration buffer consisted of 20 mM PB, 0.15 M NaCl (pH = 7.0), and 100% 0.1 M Gly-HCl (pH = 3.0) for elution. 10% (v / v) 1 M NaAc (pH = 6.5) was added to the eluent beforehand. After neutralization, turbidity appeared. After centrifugation, the supernatant was collected to precipitate the non-target protein. The 100% elution supernatant was diluted to a conductivity of 4 mS / cm and loaded onto a DSP (Borglon Biotechnology Co., Ltd.) chromatography column. The residence time on the column was 2 min. The equilibration buffer was 20 mM NaAc-HAc (pH 6.0), and the eluent was 500 mM NaCl + 20 mM NaAc-HAc (pH 6.0). Elutions were performed at 5%, 10%, and 100%, respectively. Excess potassium chains and potassium chain dimers were removed by flow-through and 5% elution, yielding a 10% eluent fraction, which was the target protein. Purity was determined using SDS-PAGE and SEC-HPLC-UV analysis. Samples with SDS-PAGE purity ≥ 95% and SEC purity ≥ 95% were obtained.

[0279] Example 13 Binding activity of CAIX / CD70 bispecific antibody against CAIX single-positive cells

[0280] Logarithmic growth phase CHOK1Q-CAIX cells were collected using 96-well U-bottom plates, centrifuged, resuspended in complete culture medium, and counted. Cells were then cultured at 2 × 10⁻⁶ cells / well using complete culture medium. 6 Seeds were prepared in 96-well U-shaped plates at 100 μL / well and culture medium was discarded after centrifugation at 300×g for 5 min. The cells were then washed once with 2% FACS buffer. The test samples were diluted to 200 nM with 2% FACS buffer, followed by 5-fold serial dilutions. Each protein dilution was added to the cells at 100 μL / well and mixed thoroughly. The cells were incubated at 4°C for 60 min. After centrifugation at 300×g for 5 min, the primary antibody was discarded, and the cells were washed once with 2% FACS buffer. The secondary antibody, Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody (DyLight), was then used. TM Resuspend the antibody in 650 μL (1:300 dilution) and incubate at 4°C for 30 min. Wash three times with 2% FACS, and resuspend each well in 200 μL. Flow cytometry (Agilent 2060R) was used to detect antibody binding rate and average fluorescence intensity, yielding the affinity of each protein for human CAIX overexpressing cells CHOK1Q-CAIX. The experimental results are shown in Figure 9A. The IgG1 isotype, representing the constant region backbone sequence of immunoglobulin IgG1, has no target specificity and serves as an isotype control for the antibody, excluding interference from non-specific binding.

[0281] As shown in Figure 9A, Girentuximab exhibited the strongest binding activity. The binding activity of Anti-CAIX-hu13E6V2-2D1-Fc was similar to that of DR31503, while the activity of DR31506 was significantly lower than that of Girentuximab, DR31503, and hu13E6V2-2D1-Fc. Vorsetuzumab showed no significant binding activity with CHOK1Q-CAIX cells. DR31506's lower affinity for CAIX allows it to effectively bind to CAIX-overexpressing cancer cells while exhibiting lower affinity for normal tissues, thus contributing to reduced toxicity.

[0282] Following the above method, the binding activity of DR31507 to CHOK1Q-CAIX cells was tested in another batch of experiments, and it was similar to that of DR31506 to CHOK1Q-CAIX cells. The experimental results are shown in Figure 9B.

[0283] Example 14 Binding activity of CAIX / CD70 bispecific antibody against CD70 single-positive cells

[0284] The cell line was replaced with CD70-positive CHOK1-CD70 cells, and the antibody binding activity with CHOK1Q-CD70 cells was detected according to the method in Example 13. The CHOK1-CD70 stable cell line was transfected with the CD70 sequence and inserted into the pcDNA3.4 vector to form the pcDNA3.4-CD70 expression plasmid. Then, the pcDNA3.4-CD70 plasmid was electrotransfected into CHOK1 cells, followed by G-418 pressure selection and single-clone acquisition.

[0285] The experimental results are shown in Figure 10A. DR31503 and DR31506 showed similar affinities for CD70, approximately 10 times lower than Vorsetumumab. aCAIX-hu13E6-2D1-Fc and Girentuximab showed no significant binding activity to CHOK1Q-CD70. The lower affinity of DR31503 and DR31506 for CD70 allows them to effectively bind to CD70-overexpressing cancer cells, while their lower affinity for normal CD70-positive cells helps reduce toxicity.

[0286] Following the above method, the binding activity of DR31507 to CHOK1Q-CD70 cells was tested in another batch of experiments, and it was similar to that of DR31506 to CHOK1Q-CD70 cells. The experimental results are shown in Figure 10B.

[0287] Example 15 Binding activity of CAIX / CD70 bispecific antibody to CAIX / CD70 double-positive cells

[0288] The cell line was replaced with CAIX / CD70 double-positive cells 786-O-CAIX, and the binding activity of DR31503, DR31506, Girentuximab, Vorsetumumab, and aCAIX-hu13E6-2D1-Fc to CAIX / CD70 double-positive renal cell carcinoma 786-O-CAIX cells was detected according to the method in Example 13. 786-O-CAIX cells were obtained by infecting 786-O cells with a recombinant lentivirus expressing human CAIX, followed by selection with puromycin.

[0289] As shown in Figure 11A, DR31503, DR31506, Girentuximab analog, and aCAIX-hu13E6V2-2D1-Fc exhibited similar and strong binding activity with double-positive cells, while Vorsetumumab analog showed relatively weak binding activity. This is related to the high expression of CAIX in 786-O-CAIX cells.

[0290] Following the above method, the binding activity of DR31507 to 786-O-CAIX cells was tested in another batch of experiments, and it was similar to the binding activity of DR31506 to 786-O-CAIX cells. The experimental results are shown in Figure 11B.

[0291] Example 16 Serum stability of CAIX / CD70 bispecific antibody

[0292] Dilute the test sample with human serum to a final protein concentration of approximately 0.1 mg / mL. Filter sterilize and aliquot into sterile centrifuge tubes. Incubate at 37°C. Take samples at 0, 3, and 7 days for ELISA to detect relative activity. Coat plates with Human CD70-TS-His (100 ng / well) or CAIX-TS-His (100 ng / well) and incubate overnight at 4°C. Wash 5 times with PBST after incubation. Block with 5% skim milk powder at 37°C for 1 hour. Wash 5 times with PBST after incubation. Add protein sample diluted with 1% BSA and block at 37°C for 2 hours. Wash 5 times with PBST after incubation. Add Goat Anti-Human IgG Fc Antibody (HRP) and block at 37°C for 1 hour. Wash 5 times with PBST after incubation. Add TMB substrate and incubate at 37°C for 15 minutes for color development. Stop the reaction with 1M sulfuric acid and read the OD at 450 nm.

[0293] The results are shown in Figures 12A-12B. DR31503 and DR31506 were relatively stable in human serum at 0 days, 3 days and 7 days.

[0294] Example 17 Preparation of Antibody-Drug Conjugates

[0295] Based on the CAIX / CD70 bispecific antibody of this application, ADCs conjugated with different cytotoxic drugs were designed and synthesized, and the specific preparation is as follows.

[0296] 17.1 Fabrication of ADCs Coupled with Exatecan

[0297] Where Ab is an anti-CAIX / CD70 bispecific antibody, such as the aforementioned DR31503, DR31506, and DR31507. n represents the average DAR value, which can be a number between 1-20, 1-10, 4-8, or 5-7.

[0298] Take 10 mL of anti-CAIX / CD70 bispecific antibody (using DR31506 as an example), with a concentration of 8.8 mg / mL, and ultrafilter it into PBS (pH 7.4) solution. The buffer exchange rate should be more than 100 times, resulting in a sample concentration of 1.0 mg / mL and a volume of 64.5 mL. Add 20 eq. (relative to the molar ratio of the antibody) of TCEP (tris(2-carboxyethyl)phosphine, 10 mM TCEP solution, 1.153 mL), mix well, and incubate at 37°C for 2 h. Add 5.873 mL of DMSO to the above solution system, mix well, and then add 10 eq. of CPD3 (10 mM CPD3 stock solution, DMSO solution system, 576.5 μL), mix well, and react at 25°C with shaking for 0.5 h. Finally, add 20 eq. of cysteine ​​(10 mM cysteine ​​stock solution, 1.153 mL) to terminate the reaction. Finally, the ultrafiltration buffer of the coupled sample was changed 1000 times to the solution (20mM NaAc + 8% trehalose, pH 5.0) to obtain 62.1 mg of the ADC product DR31506-pym-PEG8-VA-PAB-Exatecan (abbreviated as 31506-CPD3; other bispecific antibodies are named in accordance with the naming of ADCs prepared in this example). 31506-CPD3 was analyzed by SEC-HPLC, and the results are shown in Figure 13A, indicating that 31506-CPD3 showed no obvious aggregates. The experimental method for SEC-HPLC is as follows:

[0299] Instrument: Agilent 1100HPLC

[0300] Column: Cytiva Superdex 200Increase 5 / 150GL

[0301] Mobile phase: 150 mM PB + 5% isopropanol, pH 7.0

[0302] Other: (1) Detection wavelength: 214nm; (2) Column temperature: 35℃; (3) Flow rate: 0.2mL / min; (4) Injector temperature: 10℃; (5) Isocratic run time: 20min.

[0303] To determine the average DAR value of 31506-CPD3, 31506-CPD3 was analyzed by RP-HPLC. The results are shown in Figure 13B, and the average DAR value was 6.3. The experimental method for detecting the DAR value of CPD3 conjugation by RP-HPLC is as follows: 3 μL of 0.5M DTT solution was added to 30 μL of sample, and the mixture was in a metal bath at 37℃ for 30 min to reduce and open the light and heavy chains of the antibody. The following method was then used for detection:

[0304] Instrument: UHPLC

[0305] Chromatographic column: Sepax RP-1000 (2.1×150mm, 5um, 1000A)

[0306] Mobile phase: A, 0.05% TFA + water; B, 0.05% TFA + 95% acetonitrile

[0307] gradient:

[0308] Other: (1) Detection wavelength: 280nm; 370nm; (2) Column temperature: 80℃; (3) Flow rate: 0.5mL / min; (4) Injector temperature: 10℃.

[0309] The DAR value calculation parameters in this embodiment of the invention are as follows: D0% represents the proportion of antibody-drug conjugates with 0 cytotoxic drugs conjugated to an antibody via a linker; D1% represents the proportion of antibody-drug conjugates with 1 cytotoxic drug conjugate ... L0% represents the proportion of the antibody light chain with 0 cytotoxic drugs conjugated via linkers to the total light chain; L1% represents the proportion of the antibody light chain with 1 cytotoxic drug conjugated via linkers to the total light chain; H0% represents the proportion of the antibody heavy chain with 0 cytotoxic drugs conjugated via linkers to that heavy chain; H1% represents the proportion of the antibody heavy chain with 1 cytotoxic drug conjugated via linkers to that heavy chain; H2% represents the proportion of the antibody heavy chain with 2 cytotoxic drugs conjugated via linkers to that heavy chain; H3% represents the proportion of the antibody heavy chain with 3 cytotoxic drugs conjugated via linkers to that heavy chain; and H4% represents the proportion of the antibody heavy chain with 4 cytotoxic drugs conjugated via linkers to that heavy chain. The average DAR value = L1% + Left H1% + Left H2% * 2 + Right H1% + Right H2% * 2 + Right H3% * 3 + Right H4% * 4.

[0310] 31503-CPD3 was prepared using the same method as 31506-CPD3 and was detected using the same method. The SEC-HPLC results of the coupled sample before purification are shown in Figure 14A, showing no obvious aggregation. The RP-HPLC results of the purified 31503-CPD3 are shown in Figure 14B, with an average DAR value of 6.0 determined by RP-HPLC.

[0311] 31507-CPD3 was prepared using the same method as 31506-CPD3 and was detected using the same method. The SEC-HPLC results for 31507-CPD3 are shown in Figure 15A, showing no obvious aggregation. The RP-HPLC results are shown in Figure 15B, with an average DAR value of 6.1 for 31507-CPD3 determined by RP-HPLC.

[0312] 17.2 Fabrication of ADCs Coupled with Dxd

[0313] Where Ab is an anti-CAIX / CD70 bispecific antibody, such as the aforementioned DR31503, DR31506, and DR31507. n represents the average DAR value, which can be a number between 1-20, 1-10, 4-8, or 5-7.

[0314] Taking DR31506 as an example, compound MC-GGFG-Dxd was used instead of compound CPD3 in Example 17.1 to obtain the conjugate product DR31506-MC-GGFG-Dxd (abbreviated as 31506-Dxd; other bispecific antibodies are named according to the abbreviation of the ADC prepared in this example). 31506-Dxd was detected by SEC-HPLC, and the results are shown in Figure 16A, indicating no obvious aggregates in 31506-Dxd. The SEC-HPLC detection method is the same as the detection method for 31506-CPD3 in Example 17.1.

[0315] To determine the average DAR value of 31506-Dxd, 31506-Dxd was analyzed by RP-HPLC. The results are shown in Figure 16B, and the average DAR value was 6.1. The experimental method for RP-HPLC detection of the DAR value of Dxd coupling was as follows: 3 μL of 0.5M DTT solution was added to 30 μL of sample, and the mixture was incubated in a metal bath at 37℃ for 30 min to reduce and open the light and heavy chains of the antibody. Then, RP detection was performed using the following method:

[0316] Instrument: UHPLC

[0317] Column: Agilent PLRP-S2.1×100mm, 5µm

[0318] Mobile phase: A, 0.1% TFA + water; B, 0.1% TFA + 95% acetonitrile

[0319] gradient:

[0320] Other: (1) Detection wavelength: 280nm; 370nm; (2) Column temperature: 70℃; (3) Flow rate: 0.4mL / min; (4) Injector temperature: 10℃.

[0321] 31503-Dxd can be prepared using the same method as 31506-Dxd, and the same detection method can be used for detection. The detection results of SEC-HPLC are shown in Figure 17A; the detection results of RP-HPLC are shown in Figure 17B. The average DAR value of 31503-Dxd determined by RP-HPLC is 5.3.

[0322] Similarly, 31507-Dxd can be prepared by referring to the preparation method of 31506-Dxd.

[0323] 17.3 Fabrication of ADCs Coupled with MMAE

[0324] Where Ab is an anti-CAIX / CD70 bispecific antibody, such as the aforementioned DR31503, DR31506, and DR31507. n represents the average DAR value, which can be a number between 1-20, 1-10, 2-8, or 3-7.

[0325] Taking DR31506 as an example, compound MC-VC-PAB-MMAE was used instead of compound CPD3 in Example 17.1, and the amount of 10mM TCEP was adjusted from 20 eq to 5 eq, and the coupling reaction temperature was adjusted from 25℃ to 10℃, resulting in the conjugate product DR31506-MC-VC-PAB-MMAE (abbreviated as 31506-VCMMAE; other bispecific antibodies are named according to the abbreviation of the ADC prepared in this example). 31506-VCMMAE was detected by SEC-HPLC, and the results are shown in Figure 18A, indicating no obvious aggregates in 31506-VCMMAE. The SEC-HPLC detection method is the same as the detection method for 31506-CPD3 in Example 17.1.

[0326] To determine the average DAR value of 31506-VCMMAE, HIC detection was performed on 31506-VCMMAE. The detection results are shown in Figure 18B, and the average DAR value was 3.4. The experimental method for HIC detection of the VCMMAE-coupled DAR value is as follows:

[0327] Instrument: HPLC

[0328] Chromatographic column: TSKgel Butyl-NPR: 4.6mm*3.5cm, 2.5μm

[0329] Mobile phase:

[0330] A: 50mM phosphate, 1.5M (NH4)2SO4, pH 7.0 / isopropanol (95:5v / v)

[0331] B: 50mM phosphate, pH 7.0 / isopropanol (80:20 v / v)

[0332] gradient:

[0333] Other parameters: Flow rate: 0.8 mL / min, column temperature: 30℃, wavelength: 280 nm; 248 nm.

[0334] 31503-VCMMAE was prepared using the same method as 31506-VCMMAE and was detected using the same method. The SEC-HPLC results are shown in Figure 19A; the HIC results are shown in Figure 19B. The average DAR value of 31503-VCMMAE determined by the HIC method was 5.2.

[0335] Similarly, 31507-VCMMAE can be prepared by referring to the preparation method of 31506-VCMMAE.

[0336] Example 18: Killing activity of antibody-drug conjugates against tumor cells

[0337] 18.1 In vitro killing activity of DR31503-VCMMAE against OSRC-2-CAIX cells

[0338] OSRC-2-CAIX cells were obtained by infecting the OSRC-2 renal cell carcinoma cell line with a recombinant lentivirus expressing human CAIX, followed by selection with puromycin. The obtained cell line simultaneously highly expressed CD70 and CAIX. Logarithmically growing OSRC-2-CAIX cells were trypsinized, resuspended in complete culture medium (+10% FBS + 1% PS + 1 μg / mL Puromycin), counted, and adjusted to a cell density of 20,000 cells / mL. 100 μL / well was added to a 96-well plate and incubated overnight at 37°C. The next day, the sample was diluted to the specified concentration with culture medium (containing 10% FBS) (starting concentration 500 nM, prepared concentration x 2, 5-fold serial dilution). 45 μL of liquid was aspirated from the overnight incubation plate, and 50 μL of the diluted sample was added to a 96-well plate. After incubation for 120 h, cell viability was detected using the CellTiter-Glo luminescent cell viability assay kit (Promega). The experimental results are shown in Figure 20. The maximum killing effect of DR31503-VCMMAE showed a dose-dependent pattern. With increasing dose, the maximum killing effect of DR31503-VCMMAE approached ~50%. In terms of killing effect, DR31503-VCMMAE > Vorsetuzumab-VCMMAE. IgG1 isotype-VCMMAE served as a non-targeted isotype control of DR31503-VCMMAE, with the antibody replaced by IgG1 isotype (Zhixiang). The preparation of IgG1 isotype-VCMMAE followed the instructions in Example 17.3.

[0339] 18.2 In vitro killing activity of DR31503-Dxd and DR31506-Dxd against OSRC-2-CAIX cells

[0340] OSRC-2-CAIX was used as the target cell line, with 2000 cells / well seeded in 96-well plates. The test samples were diluted to the target concentration, incubated for 144 h, and then detected using the CellTiter-Glo assay kit. The experimental results are shown in Figure 21. DR31503-Dxd and DR31506-Dxd showed maximum killing effects of 43% and 49% on OSRC-2-CAIX, respectively, which were superior to Vorsetuzumab-Dxd.

[0341] 18.3 In vitro killing activity of DR31506-CPD3 against OSRC-2-CAIX cells

[0342] OSRC-2-CAIX was used as the target cell line, seeded at 2000 cells / well in 96-well plates. The test samples were diluted to the target concentration, incubated for 144 h, and then analyzed using a CellTiter-Glo assay kit. The experimental results are shown in Figure 22. The results indicate that DR31506-CPD3 achieved a maximum killing effect of 61% on OSRC-2-CAIX, with an IC50 concentration of [missing value]. 50 The concentration is 1.56 nM, and its cytotoxic activity is superior to Vorsetumumab-CPD3 (IC). 50 (83.59 nM) and Girentuximab-CPD3 (IC 50 (5.141 nM).

[0343] 18.4 In vitro killing activity of DR31506-CPD3 and DR31507-CPD3 against 786-O cells or 786-O-CAIX cells

[0344] 786-O is a renal cell carcinoma cell line with high CD70 expression and low CAIX expression. To verify the effect of CAIX expression level on the cytotoxic activity of DR31506- and DR31507-CPD3, the in vitro cytotoxic activity of DR31506-CPD3, DR31507-CPD3, and their controls against 786-O or 786-O-CAIX cells was detected. 786-O / 786-O-CAIX cells were used as target cells, seeded at 2000 cells / well in 96-well plates, and the test samples were diluted to the target concentration. After incubation for 144 h, the results were analyzed using a CellTiter-Glo assay kit. The results are shown in Figures 23A-23B. For 786-O wild-type cells, the killing activities of DR31506-CPD3 and DR31507-CPD3 were similar and weaker than those of Vorsetumumab-CPD3. For 786-O-CAIX cells overexpressing CAIX, the killing activities of DR31506-CPD3 and DR31507-CPD3 were significantly enhanced and significantly better than those of Vorsetumumab-CPD3, suggesting that high expression of CAIX can enhance the killing activities of DR31506- and DR31507-CPD3. In this embodiment, DR31506 isotype1 is a mono-Girentuximab reference product. Its first heavy chain (SEQ ID NO. 91) is the C-terminus of the anti-RSV VHH linked to the human IgG1 Fc via a hinge region. The second heavy chain (SEQ ID NO. 92) is the C-terminus of the Girentuximab VH linked to the constant region of the human IgG1 heavy chain (CH1 + hinge region + Fc). The light chain (SEQ ID NO. 64) is the light chain of Girentuximab. DR31506 isotype 2 is a reference mono-Vorsetumumab. Its first heavy chain (SEQ ID NO. 91) is the C-terminus of the anti-RSV VHH linked to the human IgG1 Fc via a hinge region. The second heavy chain (SEQ ID NO. 89) is the C-terminus of the VH of Vorsetumumab linked to the constant region of the human IgG1 heavy chain (CH1 + hinge region + Fc). The light chain (SEQ ID NO. 66) is the light chain of Vorsetumumab. DR31506 isotype 1-CDP3 and DR31506 isotype 2-CPD3 were prepared according to Example 17.1.

[0345] 18.5 In vitro killing activity of DR31506-CPD3 and DR31507-CPD3 against OSRC-2 cells or OSRC-2-CAIX cells

[0346] OSRC-2 is a renal cell carcinoma line with high CD70 expression and moderate CAIX expression. To verify the effect of CAIX expression level on the killing activity of DR31506-CPD3 and DR31507-CPD3, the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, and their controls against OSRC-2 and OSRC-2-CAIX cells was examined. OSRC-2 / OSRC-2-CAIX cells were used as target cells, seeded at 2000 cells / well in 96-well plates, and the test samples were diluted to the target concentration. After incubation for 144 h, the results were analyzed using a CellTiter-Glo assay kit. As shown in Figures 24A-24B, in the OSRC-2 wild-type cell line, the cytotoxic activity of DR31506-CPD3 was slightly better than that of DR31507-CPD3 and Vorsetumumab-CPD3, while the latter two had comparable activities. However, in OSRC-2-CAIX cells overexpressing CAIX, the cytotoxic activities of DR31506-CPD3 and DR31507-CPD3 were comparable, but significantly better than Vorsetumumab-CPD3, further demonstrating that high expression of CAIX can enhance the cytotoxic activity of DR31506- and DR31507-CPD3.

[0347] 18.6 In vitro killing activity of DR31506-CPD3 and DR31507-CPD3 against HT29-CAIX cells

[0348] HT-29-CAIX is a colon cancer cell line with low CD70 expression and high CAIX expression. To verify the effect of CD70 expression level on the killing activity of DR31506-CPD3 and DR31507-CPD3, the in vitro killing activity of DR31506-CPD3, DR31507-CPD3, and their controls against HT-29-CAIX was detected. HT-29-CAIX was used as the target cell line, and 2000 cells / well were seeded in 96-well plates. The test samples were diluted to the target concentration, incubated for 144 h, and then detected using a CellTiter-Glo assay kit. The experimental results are shown in Figure 25. DR31506-CPD3 and DR31507-CPD3 were significantly superior to Vorsetumumab-CPD3 in killing HT-29-CAIX cells. In comparison, DR31506-CPD3 and DR31507-CPD3 showed relatively weak killing effect on HT-29-CAIX cells, suggesting that low expression of CD70 reduces the effective killing activity of DR31506-CPD3 and DR31507-CPD3 to some extent.

[0349] Example 19: In vivo antitumor effects of antibody-drug conjugates with different loadings in a Balb / c nude mouse OSRC-2-CAIX xenograft model.

[0350] OSRC-2-CAIX cells (human renal cell carcinoma line) overexpressing CAIX were subcutaneously inoculated into the right flank of SPF-grade Balb / cnude mice to establish an OSRC-2-CAIX tumor-bearing mouse model. Four days after inoculation, tumor volume was measured in each mouse, and the mice were divided into five groups (n=6 per group). On the day of grouping (D0), the mice were administered the drug according to their group (tail vein injection, single dose). The grouping and administration regimens are shown in Table 11. Tumor volume and body weight were monitored 2–3 times per week after administration until day 25. Because OSRC-2-CAIX easily induces weight loss in mice, the relative tumor inhibition rate (TGI%) was calculated on day 22 after grouping using the following formula: TGI% = 100% * [1 - (tumor volume in treatment group – tumor volume in treatment group before administration) / (tumor volume in control group – tumor volume in control group before administration)].

[0351] Table 11

[0352] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 26 and Table 11. On day 22 after grouping, compared with the Vehicle group, the tumor inhibition rates of the Vorsetuzumab analog-VCMMAE group, DR31503-VCMMAE group, DR31503-Dxd group, and DR31503-CPD3 group were 19.33%, 53.76%, 84.95%, and 106.04%, respectively. DR31503-VCMMAE, DR31503-Dxd, and DR31503-CPD3 all showed significant anti-tumor effects in the OSRC-2-CAIX tumor-bearing model, and all three were superior to Vorsetuzumab analog-VCMMAE. Simultaneously, mouse body weight was measured. On day 22 after grouping, there were no significant changes in body weight in the DR31503-VCMMAE group, DR31503-Dxd group, and DR31503-CPD3 group.

[0353] Example 20: In vivo antitumor effects of antibody-drug conjugates with different loadings in a Balb / c nude mouse OSRC-2-CAIX xenograft model.

[0354] OSRC-2-CAIX cells (human renal cell carcinoma line) overexpressing CAIX were subcutaneously injected into the right flank of SPF-grade Balb / cnude mice to establish an OSRC-2-CAIX tumor-bearing mouse model. Three days after tumor cell inoculation, tumor volume was measured in each mouse, and the mice were divided into five groups (n=7 per group). On the day of grouping (D0), the mice were administered the drug according to their group (tail vein injection, single dose), as shown in Table 12. Tumor volume and body weight were monitored 2–3 times per week after drug administration until day 24. Because OSRC-2-CAIX readily induces weight loss in mice, the relative tumor inhibition rate (TGI%) was calculated on day 20 after grouping.

[0355] Table 12

[0356] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 27 and Table 12. On day 22 after grouping, compared with the Vehicle group, the tumor inhibition rates of the Vorsetuzumab analog-CPD3 group, the combination of Girentuximab analog-CPD3 and Vorsetuzumab analog-CPD3, the low-dose DR31506-CPD3 group, and the high-dose DR31506-CPD3 group were 7.68%, 79.70%, 66.49%, and 92.15%, respectively. DR31506-CPD3 showed significant dose-dependent antitumor effects in the OSRC-2-CAIX tumor-bearing model, and its antitumor efficacy was superior to that of an equivalent loading amount (equimolar concentration loading) of Vorsetuzumab analog-CPD3. There was no significant difference between DR31506-CPD3 and its combination with equivalent loading amounts of Girentuximab analog-CPD3 and Vorsetuzumab analog-CPD3. Meanwhile, the body weight of the mice was measured. On day 20, the body weight of the Vehicle group and the Vorsetuzumab analog-CPD3 group decreased significantly, while the body weight of the other groups did not change significantly.

[0357] Example 21 Antitumor efficacy of antibody-drug conjugate in Balb / c nude mouse HT-29-CAIX xenograft tumor model

[0358] A mouse model of HT-29-CAIX tumor bearing was established by subcutaneously inoculating SPF-grade Balb / c nude mice into the right flank. Eight days after inoculation, tumor volume was measured in each mouse, and the mice were divided into six groups (n=5 per group). On the day of grouping (D0), the mice were administered the drug according to their group (tail vein injection, QW×3). The grouping and administration regimens are shown in the table. Tumor volume and body weight were monitored 2–3 times per week after drug administration until day 21, and the relative tumor inhibition rate (TGI) was calculated.

[0359] Table 13

[0360] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 28 and Table 13. On day 21 after grouping, compared with the Vehicle group, the tumor inhibition rates of the Vorsetuzumab analog-CPD3 group, the DR31506 isotype1-CPD3 and DR31506 isotype2-CPD3 combination group, the low-dose and high-dose DR31506-CPD3 groups, and the low-dose DR31507-CPD3 group were 17.55%, 53.49%, 66.07%, 78.74%, and 62.93%, respectively. DR31506-CPD3 and DR31507-CPD3 showed significant dose-dependent antitumor effects in the HT-29-CAIX tumor-bearing model (G4 / G5 / G6 vs. G1, P<0.001), and their antitumor efficacy was comparable to that of G4 (G6 vs. G4, P>0.05), which was superior to the equivalent loading (equimolar concentration loading) of Vorsetuzumab analog-CPD3 group and the combination of DR31506 isotype1-CPD3 and DR31506 isotype2-CPD3 (G4 / G6 vs. G2, P<0.001; G4 / G6 vs. G3, P<0.001).

[0361] Example 22 Antitumor efficacy of antibody-drug conjugate in B-NDG mouse 786-O-CAIX xenograft tumor model

[0362] The 786-O-CAIX xenograft tumor was cut into small pieces approximately 2mm × 2mm × 2mm in size and injected subcutaneously into the right flank of B-NDG mice using a tumor inoculation needle to establish a 786-O-CAIX tumor-bearing mouse model. Fourteen days after tumor inoculation, the tumor volume of each mouse was measured, and the mice were divided into four groups (n=5 per group). On the day of grouping (D0), the mice were administered the drug according to their group (tail vein injection, D0 / D11). The grouping and administration regimens are shown in the table. Tumor volume and body weight were monitored 2–3 times per week after drug administration until D22, and the relative tumor inhibition rate (TGI) was calculated.

[0363] Table 14 Summary of Experimental Design and Results Note: All statistical data are expressed as Mean ± SE. Data analysis was performed using Two-way ANOVA. iv: Tail vein injection.

[0364] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 29A and Table 14. On day 22 after grouping, compared with the Vehicle group, the tumor inhibition rates of the DR31506 isotype1-CPD3 and DR31506 isotype2-CPD3 combination group, the DR31506-CPD3 group, and the DR31507-CPD3 group were 48.82%, 93.10%, and 97.84%, respectively. DR31506-CPD3 and DR31507-CPD3 showed significant antitumor activity in the 786-O-CAIX tumor-bearing model (G3 / G4 vs. G1, P<0.001), and their antitumor efficacy was comparable (G3 vs. G4, P>0.05), superior to the combined use of DR31506 isotype1-CPD3 and DR31506 isotype2-CPD3 with equivalent loading (equimolar concentration loading) (G3 / G4 vs. G2, P<0.001). During the experiment, mouse body weight was monitored, as shown in Figure 29B and Table 14; no significant changes in body weight were observed in any group.

[0365] Example 23 Antitumor efficacy of antibody-drug conjugate in B-NDG mouse 786-O-CAIX xenograft tumor model

[0366] 786-O-CAIX xenograft tumors were cut into small pieces approximately 2mm × 2mm × 2mm in size and injected subcutaneously into the right flank of B-NDG mice using a tumor inoculation needle to establish a 786-O-CAIX tumor-bearing mouse model. Nine days after tumor inoculation, the tumor volume of each mouse was measured, and the mice were divided into three groups (n=5 per group). On the day of grouping (D0), the mice were administered the drug according to their group (tail vein injection, QW×3). The grouping and administration regimens are shown in the table. Tumor volume and body weight were monitored 2–3 times per week after drug administration until D22, and the relative tumor inhibition rate (TGI) was calculated.

[0367] Table 15 Summary of Experimental Design and Results Note: All statistical data are expressed as Mean ± SE. Data analysis was performed using Two-way ANOVA. iv: Tail vein injection.

[0368] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 30A and Table 15. On day 22 after grouping, compared with the Vehicle group, the tumor inhibition rates of the low-dose and high-dose DR31507-CPD3 groups were 47.15% and 93.18%, respectively. DR31507-CPD3 showed significant dose-dependent antitumor activity in the 786-O-CAIX tumor-bearing model (G2 / G3 vs. G1, P<0.001). During the experiment, the body weight of mice was monitored, as shown in Figure 30B and Table 15. There were no significant changes in body weight among the groups.

[0369] Example 24 Antitumor efficacy of antibody-drug conjugate in Balb / c nude mouse renal cell carcinoma PDX model LD1-2046-362511

[0370] The successfully resuscitated FP1+2 generation LD1-2046-362511 human renal cell carcinoma xenografts were cut into small pieces approximately 3mm × 3mm × 3mm in size and inoculated subcutaneously into the right back of Balb / c nude mice using a tumor inoculation needle (efficacy experiment generation: FP1+3). Mice were observed after inoculation, and tumor growth was monitored. On day 17 after inoculation, mice were divided into three groups (n=4 per group), and the drugs were administered according to group assignment (D0) via tail vein injection, Q2W×2. The grouping and administration regimens are shown in the table. Tumor volume and body weight were monitored 2–3 times per week after administration until day 38. Since the Vehicle group underwent euthanasia on day 21 when the tumor volume exceeded 3000 mm^3, the relative tumor inhibition rate (TGI) was calculated based on day 21.

[0371] Table 16 Summary of Experimental Design and Results

[0372] All statistical data are expressed as Mean ± SE. Data analysis was performed using two-way ANOVA. iv: Tail vein injection.

[0373] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 31A and Table 16. On day 21 after grouping, compared with the Vehicle group, the tumor inhibition rates of the Vorsetuzumab analog-CPD3 group and the DR31507-CPD3 group were 89.52% and 97.42%, respectively. DR31507-CPD3 showed significant antitumor activity in the human renal cell carcinoma PDX model LD1-2046-362511 (G3 vs. G1, P<0.001), and the antitumor efficacy of DR31507-CPD3 was superior to that of an equal dose of Vorsetuzumab analog-CPD3. During the experiment, the body weight of mice was monitored, as shown in Figure 31B and Table 16. There were no significant changes in the body weight of mice in each group.

[0374] Example 25 Antitumor efficacy of antibody-drug conjugate in Balb / c nude mouse hepatocellular carcinoma PDX model LD1-2011-362539

[0375] The tumor fragments of the successfully passaged FP2+2 generation LD1-2011-362539 human hepatocellular carcinoma xenograft were cut into small pieces approximately 3mm × 3mm × 3mm in size and subcutaneously injected into the right back of BALB / c nude mice using a tumor fragment inoculation needle (the passage number for the efficacy experiment was FP2+3). Mice were observed after inoculation, and tumor growth was monitored. On day 19 after inoculation, mice were divided into four groups (n=4 per group), and the drug was administered according to the group on the day of grouping (D0) (tail vein injection, QW×4). The grouping and administration regimens are shown in the table. Tumor volume and body weight were monitored 2–3 times per week after drug administration until day 28, and the relative tumor inhibition rate (TGI) was calculated.

[0376] Table 17 Summary of Experimental Design and Results

[0377] All statistical data are expressed as Mean ± SE. Data analysis was performed using two-way ANOVA. iv: Tail vein injection.

[0378] The changes in tumor volume and tumor inhibition rate in mice are shown in Figure 32A and Table 17. On day 28 after grouping, compared with the Vehicle group, the tumor inhibition rates of the Vorsetuzumab analog-CPD3 group and the low- and high-dose DR31507-CPD3 groups were 87.47%, 99.41%, and 105.49%, respectively. DR31507-CPD3 showed significant dose-dependent antitumor activity in the human liver cancer PDX model LD1-2011-362539 (G3 / G4 vs. G1, P<0.001), and the antitumor efficacy of DR31507-CPD3 was superior to that of the same dose of Vorsetuzumab analog-2-CPD3 (G3 vs. G2, P<0.05). During the experiment, the body weight of mice was monitored, as shown in Figure 32B and Table 17. There were no significant changes in the body weight of mice in each group.

[0379] Example 26 Pharmacokinetic Study of Antibody-Drug Conjugate in Cynomolgus Monkeys

[0380] Four healthy cynomolgus macaques (half male and half female, 3–4 years old) were divided into two groups and administered DR31506-CPD3 via single intravenous infusion, either 30 mg / kg or 45 mg / kg, as shown in Table 18. Whole blood samples were collected before administration, immediately after administration, and at 2, 6, 24, 72, 120, 168, 336, and 504 hours after administration to prepare serum and plasma samples. Serum samples were used to detect the ADC and total anti-drug concentration (TAD) of DR31506-CPD3, while plasma samples were used to detect the concentration of free small molecules. Blood concentration-time curves were plotted, and pharmacokinetic parameters were calculated to evaluate the pharmacokinetic characteristics and the shedding of conjugated small molecules in the cynomolgus macaques.

[0381] Table 18. Dosage regimen for cynomolgus monkeys

[0382] The plasma concentration-time curves of DR31506-CPD3 are shown in Figure 33, and the pharmacokinetic parameters are shown in Table 19. In cynomolgus monkeys, the mean half-life of DR31506-CPD3 was approximately 4.5 days. The exposure levels of ADC and total antibody were essentially the same, with no sex difference in exposure. Exposure was positively linearly correlated with dose, and no ADA production was detected at the endpoint. The exposure levels of Exatecan in the plasma of cynomolgus monkeys were low at each dose group, and the small molecule shedding rate was [missing information]. This indicates that DR31506-CPD3 is relatively stable in cynomolgus monkeys, with a low rate of small molecule shedding.

[0383] Table 19 Pharmacokinetic parameters in cynomolgus monkeys

[0384] In summary, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0385] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-CAIX single-domain antibody comprising CDR1 to CDR3 as shown below: (1) CDR1 with an amino acid sequence as shown in SEQ ID NO.1 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity; CDR2 with an amino acid sequence as shown in SEQ ID NO.2 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity; or CDR3 with an amino acid sequence as shown in SEQ ID NO.3 or having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity; or (2) CDR1 with an amino acid sequence as shown in SEQ ID NO.4 or having 80%, 85%, 90%, 95%, 98%, or 99% or more of the sequence identity, CDR2 with an amino acid sequence as shown in SEQ ID NO.5 or having 80%, 85%, 90%, 95%, 98%, or 99% or more of the sequence identity, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6 or having 80%, 85%, 90%, 95%, 98%, or 99% or more of the sequence identity.

2. The single-domain antibody as described in claim 1, characterized in that, The single-domain antibody includes: a) An amino acid sequence as shown in any one of SEQ ID NO. 7–21; or b) An amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with one of SEQ ID NO. 7 to 21, and having the function of the amino acid sequence defined in a).

3. A fusion protein of an anti-CAIX single-domain antibody, comprising a first domain of the single-domain antibody as described in any one of claims 1 to 2, and further comprising a second domain for prolonging the in vivo half-life and / or having a binding effector effector.

4. The fusion protein as described in claim 3, characterized in that, The second domain includes one or more of the following: a serum albumin fragment, a polyethylene glycol fragment, and a single-domain antibody that binds to human serum albumin; and / or, the second domain includes an immunoglobulin Fc region; and / or, the second domain includes a molecule that has affinity for CD3 present on T cells and / or is capable of binding to CD3 present on T cells.

5. A bispecific antibody comprising the anti-CAIX single-domain antibody as described in any one of claims 1 to 2.

6. The bispecific antibody as described in claim 5, characterized in that, It also includes domains that target EGFR, MET, HER3, HER2, CD70, or Trop2.

7. The bispecific antibody as described in claim 6, characterized in that, The CD70-targeting domain is an anti-CD70 antibody or its antigen-binding fragment.

8. The bispecific antibody of claim 7, wherein the anti-CD70 antibody or its antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO. 67, HCDR2 as shown in SEQ ID NO. 68, HCDR3 as shown in SEQ ID NO. 69, LCDR1 as shown in SEQ ID NO. 70, LCDR2 as shown in SEQ ID NO. 71, and LCDR3 as shown in SEQ ID NO.

72.

9. The bispecific antibody as described in claim 8, characterized in that, The anti-CD70 antibody or its antigen-binding fragment comprises VH, which has an amino acid sequence as shown in SEQ ID NO.73 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with it, and / or VL, which has an amino acid sequence as shown in SEQ ID NO.74 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with it.

10. The bispecific antibody as described in claim 7, characterized in that, The bispecific antibody further comprises an Fc fragment, wherein the Fc fragment is selected from one or more combinations of IgG, IgA1, IgA2, IgD, IgE, and IgM; preferably, the Fc fragment is selected from the Fc of IgG1, IgG2, IgG3, or IgG4.

11. The bispecific antibody as described in claim 10, characterized in that, The bispecific antibody comprises a light chain, a first heavy chain, and a second heavy chain.

12. The bispecific antibody as described in claim 11, characterized in that, The first heavy chain contains an anti-CAIX single-domain antibody fused to the Fc fragment.

13. The bispecific antibody as described in claim 11, characterized in that, The light chain and / or the second heavy chain contains an anti-CD70 antibody or an antigen-binding fragment thereof.

14. The bispecific antibody as described in claim 11, characterized in that, The Fc segment of the first heavy chain contains a Knob mutation, and the Fc segment of the second heavy chain contains a Hole mutation, or the Fc segment of the first heavy chain contains a Hole mutation, and the Fc segment of the second heavy chain contains a Knob mutation.

15. The bispecific antibody as described in claim 11, characterized in that, The Fc fragment of the first heavy chain and / or the second heavy chain also contains the M428L mutation.

16. The bispecific antibody as described in claim 11, characterized in that, The Fc fragment has an amino acid sequence as shown in any of SEQ ID No. 76 to 83.

17. The bispecific antibody according to claim 11, characterized in that, The first heavy chain contains an amino acid sequence as shown in any one of SEQ ID No. 86 to 88, the second heavy chain contains an amino acid sequence as shown in any one of SEQ ID No. 89 to 90, and the light chain contains an amino acid sequence as shown in SEQ ID No.

66.

18. An isolated polynucleotide encoding a single-domain antibody as claimed in any one of claims 1 to 2, a fusion protein as claimed in any one of claims 3 to 4, or a bispecific antibody as claimed in any one of claims 5 to 17.

19. An expression vector containing the isolated polynucleotide as described in claim 18.

20. An expression system comprising an expression vector as described in claim 19 or a genome in which an exogenous polynucleotide as described in claim 18 is integrated.

21. A method for preparing a single-domain antibody as described in any one of claims 1 to 2, a fusion protein as described in any one of claims 3 to 4, or a bispecific antibody as described in any one of claims 5 to 17, comprising the following steps: culturing the expression system as described in claim 20 under conditions suitable for expressing the single-domain antibody, fusion protein, or bispecific antibody, thereby expressing the single-domain antibody, fusion protein, or bispecific antibody; and purifying and separating the single-domain antibody, fusion protein, or bispecific antibody.

22. An immunoconjugate or immunodrug comprising the anti-CAIX single-domain antibody of any one of claims 1 to 2, the fusion protein of any one of claims 3 to 4, or the bispecific antibody of any one of claims 5 to 17.

23. The immunoconjugate or immunodrug as described in claim 22, characterized in that, It also includes one or more combinations of cytotoxic drugs, molecular gels, PROTAC molecules, immune agonists, and radioactive isotopes; preferably, the cytotoxic drugs include, but are not limited to, tubulin inhibitors, topoisomerase inhibitors, or toxins for binding DNA; the molecular gels include, but are not limited to, pomalidomide, lenalidomide, and thalidomide; the PROTAC molecules include, but are not limited to, ARV-110, ARV-766, ARV-471, NX-2127, NX-5948, CFT1946, CFT8919, and DT2216; and the radioactive isotopes include, but are not limited to, […]. 18 F, 44 Sc、 47 Sc、 64 Cu、 67 Ga、 86 Y、 89 Zr、 90 Y、 99m Tc, 111 In、 123 I, 124 I, 131 I, 177 Lu、 211 At、 212 Pb.

24. An antibody-drug conjugate, characterized in that, Having the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: Ab-(LD) n Formula (I), wherein Ab is the anti-CAIX single-domain antibody according to any one of claims 1 to 2, the fusion protein according to any one of claims 3 to 4, or the bispecific antibody according to any one of claims 5 to 17; L is the linker arm; D is the cytotoxic drug; and n is a number between 1 and 20.

25. The antibody-drug conjugate as described in claim 24, characterized in that, D is selected from one or more of the following: maytansine derivative DM1, maytansine derivative DM4, monomethyl aurestatin E (MMAE), monomethyl aurestatin F (MMAF), duocarmycin, pyrrolobenzodiazepine (PBD), camptothecin, SN38, exatecan, Dxd, tubulysins, amanitin, PNU-159682, and calicheamicins.

26. The antibody-drug conjugate as described in claim 24, characterized in that, L is an enzyme-cleavable linker, an acid-cleavable linker, a linker that cleaves under reducing conditions, or a linker that cleaves under exogenous stimuli.

27. The antibody-drug conjugate as described in claim 24, characterized in that, L can be carbon-based, amino-based, amide-based, acyl-based, or -(PEG). m -、-(CH2) m - Containing heteroatoms -(CH2) m -, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide (Mal), maleimide hexanoyl (MC), maleimide propionyl (MP), VA (-Val-Ala-), VC (-Val-Cit-), VK (-Vla-Lys-), AF (-Ala-Phe-), GGFG (-Gly-Gly-Phe-Gly-), p-aminobenzyloxycarbonyl (PAB), N-succinimide 4-(N-maleimide methyl)-cyclohexane-1-carboxylic acid ester (SMCC), N-succinimide 4-(2-pyridinylthio)valerate (SPP), polyethylene glycol (PEG), pyrimidine, pyridine (py), methanesulfonylpyrimidinyl, methanesulfonylpyridinyl, and one or more combinations thereof, wherein each m is independently selected from an integer from 1 to 20.

28. The antibody-drug conjugate as described in claim 24, characterized in that, The L includes one or more combinations of the following structures: Each q is independently selected from integers from 1 to 20, for example, integers from 1 to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8; X is selected from -NH-, -O-, and -S-; Su are each independently selected from pentose, penturonic acid, hexose, and hexuronic acid; R c Each was independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups.

29. The antibody-drug conjugate according to any one of claims 24 to 28, characterized in that, The antibody-drug conjugate has any of the following structures: Wherein, Ab is an anti-CAIX single-domain antibody or a CAIX / CD70 bispecific antibody; n is selected from an integer or decimal between 1 and 20; preferably, n is selected from an integer or decimal between 1 and 10.

30. The antibody-drug conjugate according to any one of claims 24 to 29, characterized in that, The Ab is a CAIX / CD70 bispecific antibody, wherein the anti-CAIX single-domain antibody targeting CAIX includes: The amino acid sequence is as shown in SEQ ID NO.1 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.2 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.3 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3. or The amino acid sequence is as shown in SEQ ID NO.4 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR1; the amino acid sequence is as shown in SEQ ID NO.5 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR2; the amino acid sequence is as shown in SEQ ID NO.6 or has 80%, 85%, 90%, 95%, 98%, or 99% or more sequence identity with CDR3.

31. The antibody-drug conjugate as described in claim 30, characterized in that... The CAIX / CD70 bispecific antibody comprises: The first heavy chain shown in SEQ ID No. 86, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or The first heavy chain shown in SEQ ID No. 87, the second heavy chain shown in SEQ ID No. 89, and the light chain shown in SEQ ID No. 66; or The first heavy chain shown in SEQ ID No. 88, the second heavy chain shown in SEQ ID No. 90, and the light chain shown in SEQ ID No.

66.

32. A composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 24 to 31, wherein the molar ratio (DAR) of D to Ab in the composition is an integer or a decimal between 1 and 10.

33. The composition of claim 32, wherein the antibody-drug conjugate content is greater than 50%.

34. Use of the single-domain antibody as described in any one of claims 1 to 2, the fusion protein as described in any one of claims 3 to 4, the bispecific antibody as described in any one of claims 5 to 17, the antibody-drug conjugate as described in any one of claims 24 to 31, or the pharmaceutical composition as described in claim 32 or 33 in the preparation of a medicament for the diagnosis, treatment, or prevention of CAIX-positive or CD70-positive tumors.

35. The use as described in claim 34, characterized in that, The tumor is selected from one or more of the following: renal cell carcinoma, breast cancer, lung cancer, liver cancer, colorectal cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, malignant lymphoma, pancreatic cancer, and glioblastoma.

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