WO2025247308A1 - Pharmaceutical composition of cldn18.2/4-1bb binding protein and pharmaceutical use thereof - Google Patents

Abstract

Provided are a pharmaceutical composition of a CLDN18.2/4-1BB binding protein and the pharmaceutical use thereof. Specifically, the present invention relates to a pharmaceutical composition containing a CLDN18.2/4-1BB binding protein. The pharmaceutical composition contains the CLDN18.2/4-1BB binding protein and a buffering agent.

Description

A pharmaceutical composition of CLDN18.2/4-1BB binding protein and its pharmaceutical uses. Technical Field

This disclosure relates to the field of pharmaceutical formulations, and more specifically to a pharmaceutical composition comprising CLDN18.2/4-1BB binding protein, and its pharmaceutical use.

Background Technology

The Claudin protein family is an important class of tight junction proteins, generally composed of four transmembrane domains and two extracellular loops, whose main function is to maintain normal molecular exchange between cells. Claudin18 is one of the members of the Claudin protein family and has two splice variants: Claudin18 splice variant 1 (CLDN18.1) and Claudin18 splice variant 2 (CLDN18.2). The two variants differ only in the first extracellular segment by eight amino acids. The two variants have significant differences in physiological distribution: CLDN18.1 is mainly expressed in normal lung tissue, while CLDN18.2 is mainly expressed in the stomach. In healthy tissues, CLDN18.2 is usually embedded in the gastric mucosa, making it largely inaccessible to antibodies. When cells undergo malignant transformation, intercellular junctions are disrupted, exposing CLDN18.2 to antibody-binding epitopes, making it a tumor-specific target. CLDN18.2 is significantly expressed ectopically in various epithelial tumors, including gastric cancer, esophageal cancer, and pancreatic cancer, providing a biological basis for targeted cancer therapy targeting CLDN18.2. IMAB362, an IgG1 subtype anti-CLDN18.2 antibody developed by Astellas, achieved a 63.2% response rate when used in combination with chemotherapy in patients with advanced metastatic gastric and gastroesophageal cancer, surpassing historical data from chemotherapy monotherapy (2021 ASCO). This demonstrates the clinical value of CLDN18.2 as a tumor-specific antigen.

4-1BB (CD137, TNFRSF9) is a transmembrane protein belonging to the tumor necrosis factor receptor superfamily. 4-1BB is a co-stimulatory molecule expressed on the surface of CD8+ and CD4+ T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells, and neutrophils. On T cells, 4-1BB is not constitutively expressed, but rather inducibly expressed after activation of the T cell receptor (TCR). 4-1BB is expressed on the cell surface in monomeric or dimeric form, forming a trimer upon binding to its natural ligand 4-1BBL, and signaling is transduced via TNFR-associated factor (TRAF)-2 and TRAF-1. Early signaling of 4-1BB involves K-63 polyubiquitination, activation of nuclear factor (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, leading to T cell proliferation, maturation, prolonged survival, and cytokine production. Studies have shown that antibody agonists targeting 4-1BB can promote the anti-tumor function of T cells in mice (Murillo et al., Clin Cancer Res. 2008; 14(21): 6895-906). Antibodies that activate 4-1BB can increase the expression of co-stimulatory molecules in many models, induce T cell survival and proliferation, thereby enhancing the anti-tumor immune response and causing immune cells to kill tumors. Existing 4-1BB activating antibodies include Urelumab from Bristol Myers Squibb (BMS), a human IgG4 antibody (WO2005035584); Utomilumab from Pfizer, a human IgG2 antibody (Fisher et al., Cancer Immunol. 2012; 61: 1721-1733); and ADG106 from Adagene, a human IgG4 antibody (WO2019037711A1). However, the development of anti-tumor antibodies targeting 4-1BB has not progressed smoothly. The main reason is that 4-1BB can activate not only T cells inside the tumor, but also T cells in the periphery (e.g., the liver), causing liver inflammation and leading to severe liver damage (Todd Bartkowiak et al. Clin Cancer Res; 24(5) March 1, 2018). This makes the therapeutic window of 4-1BB narrow.

Given the excellent tumor specificity and targeting of CLDN18.2, and the problems existing in current agonist antibodies targeting 4-1BB, the applicant's patent application WO2024114676 provides a bispecific antibody against CLDN18.2/4-1BB that activates 4-1BB by binding to CLDN18.2 on tumor tissue, thus avoiding hepatotoxicity caused by 4-1BB and expanding the therapeutic window of the antibody. The efficacy is further enhanced when an effector-enhanced Fc (e.g., ADCC, ADCP) is used in the bispecific antibody against CLDN18.2/4-1BB.

Antibody drugs are an important class of biological drugs. Due to their large molecular weight and complex structure, they are easily degraded and polymerized by physical or chemical factors during production, storage, and use, leading to reduced activity or even inactivation. Therefore, the development of excellent antibody formulations is of paramount importance.

Summary of the Invention

This disclosure provides a pharmaceutical composition containing CLDN18.2/4-1BB binding protein, which exhibits excellent stability.

This disclosure provides a pharmaceutical composition comprising a) CLDN18.2/4-1BB binding protein and a buffer, wherein the buffer is selected from acetate buffers, citrate buffers, and histidine buffers. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer or a histidine-acetate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer.

CLDN18.2/4-1BB binding protein

WO2024114676 relates to the CLDN18.2/4-1BB binding protein, and this application incorporates the entire contents of that patent.

In the first aspect, the CLDN18.2/4-1BB binding protein disclosed herein includes a first antigen-binding domain that specifically binds to 4-1BB and a second antigen-binding domain that specifically binds to CLDN18.2, and can simultaneously or separately specifically bind to 4-1BB and CLDN18.2.

In some implementations, when the second antigen-binding domain that specifically binds to CLDN18.2 does not bind to CLDN18.2, the first antigen-binding domain that specifically binds to 4-1BB does not activate the 4-1BB signal, or binds to the 4-1BB antigen but does not activate the 4-1BB signal.

In some embodiments, the CLDN18.2/4-1BB binding protein has enhanced effector function; for example, the CLDN18.2/4-1BB binding protein includes an effector-enhanced Fc region and/or a third antigen-binding domain that specifically binds to CD16A.

In some embodiments, the Fc region that enhances the effector function increases binding to FcγR, for example, increases binding to FcγRIIB (CD32B); increases binding to FcγRIIIA (CD16A). In other embodiments, binding is reduced, decreased, or absent with FcγRIIIB (CD16B).

In some implementations, the Fc region that enhances the effector function is to increase the binding with C1q.

In some implementations, the Fc region with enhanced effector function has a glycosylation level that is normal or higher than that of the wild-type Fc region.

In some implementations, the effector functions as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

Regarding the first antigen-binding domain that specifically binds to 4-1BB:

In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2/4-1BB binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any of the amino acid sequences shown in SEQ ID NO: 10, 18-21, wherein CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. The immunoglobulin single variable domain specifically binds to the 4-1BB antigen or fragments thereof.

In some embodiments, the immunoglobulin single variable domain comprises any one or any combination of CDR1, CDR2 and CDR3 described above.

In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are shown in SEQ ID NO: 11, 12, and 13, or in SEQ ID NO: 11, 12, and 22, respectively. These are CDRs defined according to the Kabat numbering system.

In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are shown as GFTFSSYA (SEQ ID NO: 39), INSGGEST (SEQ ID NO: 40), and AKHPLTFTIATMNDYDY (SEQ ID NO: 41), or as shown as SEQ ID NO: 39, 40, and AKHPLTYTIATMNDYDY (SEQ ID NO: 42). These are CDRs defined according to the IMGT numbering system.

In some embodiments, the amino acid sequences of the CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are as shown in GFTFSSY (SEQ ID NO: 43), NSGGES (SEQ ID NO: 44), and SEQ ID NO: 13, or as shown in SEQ ID NO: 43, 44, and 22, respectively. These are CDRs defined according to the Chothia numbering system.

In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are as shown in GFTFSSYAMS (SEQ ID NO: 45), DINSGGESTF (SEQ ID NO: 46), and SEQ ID NO: 13, or as shown in SEQ ID NO: 45, 46, and 22, respectively. These are CDRs defined according to the AbM numbering system.

In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are as shown in SSYAMS (SEQ ID NO: 47), WVSDINSGGESTF (SEQ ID NO: 48), and AKHPLTFTIATMNDYD (SEQ ID NO: 49), or as shown in SEQ ID NO: 47, 48, and AKHPLTYTIATMNDYD (SEQ ID NO: 50). These are CDRs defined according to the Contact numbering system.

In some implementations, the immunoglobulin single variable domain is humanized, affinity-matured, T-cell epitope-removed/reduced, antibody deamidation reduced, and/or antibody isomerization reduced.

In some embodiments, the immunoglobulin single variable domain is obtained by removing/reducing T-cell epitopes, which have one or more variations in one or more CDRs, resulting in a decrease in the immunogenicity of the binding protein.

In some embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-64*04, IGHV3-23*03, and/or IGHV3-74*01. In some embodiments, FR1 is derived from IGHV3-64*04, FR2 from IGHV3-23*03, and FR3 from IGHV3-74*01.

In some embodiments, the amino acid sequence of the single variable domain of the immunoglobulin is as shown in any of SEQ ID NO: 10, 18-21, or has at least 80% sequence identity with any of SEQ ID NO: 10, 18-21.

In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2/4-1BB binding protein comprises the aforementioned 4-1BB binding protein of this disclosure, or comprises anti-4-1BB antibodies or antigen-binding fragments thereof from Urelumab, Utomilumab, ADG106, and WO2005035584A, WO2019037711A, US20190055314A, WO2019014328A3, and US20210206867A. The entire contents of this disclosure incorporate the aforementioned patents.

Regarding the second antigen-binding domain that specifically binds to CLDN18.2:

In some embodiments, the second antigen-binding domain in the CLDN18.2/4-1BB binding protein that specifically binds to CLDN18.2 includes a heavy chain variable region (VH) and a light chain variable region (VL).

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 37. The CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system.

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 30, 31, and 32, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 33, 34, and 35. These are CDRs defined according to the Kabat numbering system.

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSYW (SEQ ID NO: 51), IHPNSGST (SEQ ID NO: 52), and ARLKTGNSFDY (SEQ ID NO: 53), and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in QSLLNSGNQKNY (SEQ ID NO: 54), WA, and SEQ ID NO: 35. These are CDRs defined according to the IMGT numbering system.

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSY (SEQ ID NO: 55), HPNSGS (SEQ ID NO: 56), and SEQ ID NO: 32, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 33, 34, and 35. These are CDRs as defined in the Chothia numbering system.

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in GYTFTSYWMH (SEQ ID NO: 57), MIHPNSGSTN (SEQ ID NO: 58), and SEQ ID NO: 32, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 33, 34, and 35. These are CDRs defined according to the AbM numbering system.

In some embodiments, the VH that specifically binds to the second antigen-binding domain of CLDN18.2 comprises HCDR1, HCDR2, and HCDR3 as shown in TSYWMH (SEQ ID NO: 59), WMGMIHPNSGSTN (SEQ ID NO: 60), and ARLKTGNSFD (SEQ ID NO: 61), and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in LNSGNQKNYLTWY (SEQ ID NO: 62), LLIYWASTRE (SEQ ID NO: 63), and QNAYTYPF (SEQ ID NO: 64). These are CDRs defined according to the Contact Numbering System.

In some embodiments, the VH of the second antigen-binding domain that specifically binds to CLDN18.2 comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 80% or at least 90% identity with it, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 37 or having at least 80% or at least 90% identity with it.

In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4.

In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 23 or has at least 80% or at least 90% identity with it, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 24 or has at least 80% or at least 90% identity with it.

This disclosure fully incorporates the anti-CLDN18.2 antibody in WO2020200196A, wherein the antibody or its antigen-binding fragment can serve as the second antigen-binding domain specifically binding to CLDN18.2 in this disclosure. In other embodiments, the second antigen-binding domain specifically binding to CLDN18.2 comprises the anti-CLDN18.2 antibody or its antigen-binding fragment in WO2021027850A, WO2014146672A, WO2021025177A, WO2016180782A, and WO2021254481A, and the above patents are also incorporated in full into this disclosure.

The following exemplarily provides the CLDN18.2/4-1BB binding protein of this disclosure:

In some embodiments, the first antigen-binding domain that specifically binds to 4-1BB in the CLDN18.2/4-1BB binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any of the amino acid sequences shown in SEQ ID NO: 10, 18-21, wherein CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown as in SEQ ID NO: 11, 12, and 13, or as in SEQ ID NO: 11, 12, and 22, respectively. In some specific embodiments, the amino acid sequence of the single variable domain of the immunoglobulin is as shown in any one of SEQ ID NO: 10, 18-21, or has at least 80% or at least 90% sequence identity with any one of SEQ ID NO: 10, 18-21. In some specific embodiments, the single variable domain of the immunoglobulin is a single-domain antibody or VHH.

In some embodiments, the second antigen-binding domain that specifically binds to CLDN18.2 in the CLDN18.2/4-1BB binding protein comprises VH and VL. In some specific embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO: 36, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 37. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system. In some specific embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 30, 31, and 32, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 33, 34, and 35. In some specific embodiments, the VH comprises an amino acid sequence as shown in or having at least 80% or 90% identity with SEQ ID NO: 36, and the VL comprises an amino acid sequence as shown in or having at least 80% or 90% identity with SEQ ID NO: 37. In some specific embodiments, the CLDN18.2/4-1BB binding protein specifically binding to the second antigen-binding domain of CLDN18.2 comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in or having at least 80% or 90% identity with SEQ ID NO: 23, and the light chain comprises an amino acid sequence as shown in or having at least 80% or 90% identity with SEQ ID NO: 24.

In some embodiments, the CLDN18.2/4-1BB binding protein has one or more (e.g., 2, 3, 4, 5, 6) first antigen-binding domains that specifically bind to 4-1BB, and/or one or more (e.g., 2, 3, 4) second antigen-binding domains that specifically bind to CLDN18.2. In some specific embodiments, the CLDN18.2/4-1BB binding protein has two first antigen-binding domains that specifically bind to 4-1BB and two second antigen-binding domains that specifically bind to CLDN18.2.

In some embodiments, the valence ratio of the first antigen-binding domain that specifically binds to 4-1BB to the second antigen-binding domain that specifically binds to CLDN18.2 in the CLDN18.2/4-1BB binding protein is between 6:1 and 1:3 (e.g., 4:1 to 1:2), such as 1:1, 1:2, 2:1, 1:3, or 3:1.

In some embodiments, in the CLDN18.2/4-1BB binding protein, the first antigen-binding domain that specifically binds to 4-1BB is located at the N-terminus and/or C-terminus of the second antigen-binding domain that specifically binds to CLDN18.2.

In some embodiments, the CLDN18.2/4-1BB binding protein further comprises a human immunoglobulin Fc region. For example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4.

In some specific implementations, the Fc region can cause the binding protein to form a dimer molecule.

In some specific implementations, the Fc region contains mutations that prolong the in vivo half-life, which depends on the FcRn binding affinity. Prolonged half-life can allow for a reduction in the amount of drug administered to the patient and/or a decrease in dosing frequency. For example, the Fc region may have M252Y, S254T, and/or T256E mutations.

In some specific implementations, the Fc region is an effector-enhancing Fc region, such as an ADCC, ADCP, and/or CDC-enhanced Fc region; or a fucosylation-reduced Fc region. The Fc region may have mutations, and exemplary IgG1 Fc regions with enhanced effector function include substitutions of the following or any combination thereof: S239D, S239E, S239K, F241A, V262A, V264D, V264L, V264A, V264S, D265A, D265S, D265V, F296A, Y296A, R301A, I332E, S239D/I332E, S239D/A330S/I332E, S239D/A330L/I332E, S298A/D333A/K334A, P247I/A339D, P247I/A339Q, D280H/K290S, D 280H/K290S/S298D, D280H/K290S/S298V, F243L/R292P/Y300L, F243L/R292P/Y300L/P396L, F243L/R292P/Y300L/V305I/P396L, G236A/S239D/I332E, K326A/E333A, K326W/E333S, K290E/S298G/T299A, K290N/S298G/T299A, K290E/S298G/T299A/K326E or K290N/S298G/T299A/K326E, or any combination of the above positions. In some specific embodiments, the CLDN18.2/4-1BB binding protein contains the human IgG1 Fc region with the S239D/I332E mutation.

In other specific embodiments, the Fc region may be an Fc region with reduced effector function. For example, the Fc region may have mutations, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A/L235A (IgG1); V234A/G237A (IgG2); L235A/G237A/E318A (IgG4); H268Q/V309L/A330S/A331S (IgG2); C220S/C226S/C229S/P238S (IgG1); C226S/C229S/E233P/L234V/L235A (IgG1); L234F/L235E/P331S (IgG1); or S267E/L328F (IgG1).

In this disclosure, Fc regions with enhanced effector function are selected from CLDN18.2/4-1BB binding proteins (e.g., anti-CLDN18.2/4-1BB bispecific antibodies), which are significantly superior to Fc regions with unchanged or reduced effector function.

In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 38. In other embodiments, the Fc comprises the amino acid sequence shown in any of SEQ ID NO: 14-16.

In some embodiments, the Fc region of the CLDN18.2/4-1BB binding protein includes a first subunit Fc1 and a second subunit Fc2 that can associate with each other.

In some embodiments, Fc1 and Fc2 contain amino acid mutations such that Fc1 preferentially pairs with Fc2 or preferentially forms a heterodimer compared to Fc1. In some embodiments, the mutation is located in the CH3 group of Fc1 and Fc2. In some embodiments, the amino acid mutation in Fc1 and Fc2 produces greater electrostatic complementarity than the wild type without the mutation. In some embodiments, the amino acid mutation in Fc1 and Fc2 produces greater spatial complementarity than the wild type without the mutation.

In some embodiments, in Fc1 and Fc2, for example within the CH3/CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby creating a bulge (or konb) on the surface of the CH3 domain of Fc1. One or more, preferably two or three amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby creating a depression (or holosphere) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the input residues with a larger side chain volume are phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the input residues with a smaller side chain volume are serine (S), alanine (A), valine (V), or threonine (T).

In some specific embodiments, Fc1 contains at least one or at least two amino acid mutations (mortar mutation modification) selected from T366S, L368A and Y407V, and Fc2 contains T366W (mortar mutation modification); or Fc1 contains T366W (mortar mutation modification), and Fc2 contains at least one or at least two amino acid mutations (mortar mutation modification) selected from T366S, L368A and Y407V.

In some specific implementations, Fc1 and Fc2, for example, CH3, may contain a mutation from a natural non-cysteine to a cysteine, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and S354C in Fc2.

In some specific implementations, Fc1 and Fc2, for example within the Fc1CH3/Fc2CH3 interface, contain the following amino acid mutations or combinations thereof: T366Y/Y407T; T366W/Y407A; T366Y/Y407T; T394W/F405A; T366Y/F405AT; T394W/Y407T; T366W/F405WT; T394S/Y407A; F405W/T394S; D399C/K392C; T366W/T366S/L368A/Y407V; T366W/D399C/T366S/L368A/K392C/Y407V; T366W/K392 C/T366S/D399C/L368A/Y407V; S354C/T366W/Y349C/T366S/L368A/Y407 V; Y349C/T366W/S354C/T366S/L368A/Y407V; E356C/T366W/Y349C/T366S /L368A/Y407V; Y349C/T366W/E356C/T366S/L368A/Y407V; E357C/T366W/ Y349C/T366S/L368A/Y407V; and Y349C/T366W/E357C/T366S/L368A/Y407V.

In some specific embodiments, Fc1 and Fc2 further contain amino acid mutations that enable the formation of an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). The amino acid mutations that enable the formation of the electrostatic interaction interface are selected, for example, from the following: K370E/D399K/K439D/D356K/E357K/K409D; K409D/D399K; K409E/D399K; K409E/D399R; K409D/D399R; D339K/E356K; D399K/E356K/K409D/K392D; D 399K/E356K/K409D/K439D; D399K/E357K/K409D/K370D; D399K/E356K/E357K/K409D/K 392D/K370D; D399K/E357K/K409D/K392D; K392D/K409D/D399K; and K409D/K360D/D399K.

In some implementations, Fc1 and/or Fc2 contain domains derived from different antibody subtypes, such as CH3 derived from different antibody subtypes.

Furthermore, this disclosure cites schemes in WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO2007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012058768, WO2013157954, and WO2013096291 that modify the CH3 region of the Fc region to enhance heterodimerization.

In some embodiments, the first antigen-binding domain specifically binding to 4-1BB and the second antigen-binding domain specifically binding to CLDN18.2 in the CLDN18.2/4-1BB binding protein are directly or via a linker. In some embodiments, the first antigen-binding domain specifically binding to 4-1BB is directly or via a linker to the Fc region.

In some specific embodiments, the linker includes, but is not limited to, amino acid sequences such as (GmSn)h, (GGNGT)h, (YGNGT)h, or (EPKSS)h, wherein m and n are each independently selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some specific embodiments, the linker may be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures. In some specific embodiments, the linker is a flexible linker. In some specific implementations, the connector is selected from _G4S , GS, GAP, ( _G4S ) ₂ , ( _G4S ) ₃ , ( _G4S ) ₄ , ( _G4S ) ₅ , ASGS, for example ( _G4S ) ₂ and ( _G4S ) ₃ .

In some specific embodiments, the CLDN18.2/4-1BB binding protein comprises a first polypeptide chain and a second polypeptide chain, the first and second polypeptide chains from the N-terminus to the C-terminus as follows:

(1) First polypeptide chain: [VH region of the second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 1]a-[first antigen-binding domain that specifically binds to 4-1BB];

Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-CL

(2) First polypeptide chain: [first antigen-binding domain that specifically binds to 4-1BB]-[linker 1]a-[VH of the second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region;

Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of CLDN18.2]-CL

(3) First polypeptide chain: [VH]-CH1-Fc region of the second antigen-binding domain that specifically binds to CLDN18.2;

Second polypeptide chain: [specifically binds to the first antigen-binding domain of 4-1BB]-[linker 1]a-[specifically binds to the second antigen-binding domain of CLDN18.2]-CL

(4) First polypeptide chain: [VH]-CH1-Fc region of the second antigen-binding domain that specifically binds to CLDN18.2;

Second polypeptide chain: [VL specifically binds to the second antigen-binding domain of CLDN18.2]-[linker 1]a-CL [specifically binds to the first antigen-binding domain of 4-1BB]

(5) First polypeptide chain: [VH of the second antigen-binding domain that specifically binds to CLDN18.2]-CH1-Fc region-[linker 2]b-[the first antigen-binding domain that specifically binds to 4-1BB];

Second polypeptide chain: [VL specifically binds to the second antigen-binding domain of CLDN18.2]-[linker 1]a-CL [specifically binds to the first antigen-binding domain of 4-1BB]

Wherein, - represents a peptide bond, the linker is a polypeptide capable of linking, and linkers 1, 2, 3, and 4 can be the same or different; a, b, c, and d can be independently selected from 1 or 0, for example, a, b, c, and d are all 1. The linkers are independently selected, for example, from _G4S , GS, GAP, ( _G4S ) ₂ , ( _G4S ) ₃ , ( _G4S ) ₄ , ( _G4S ) ₅ , and ASGS, for example, ( _G4S ) ₂ and ( _G4S ) ₃ .

In some embodiments, a CLDN18.2/4-1BB binding protein is provided, comprising first and second polypeptide chains. In some specific embodiments, the amino acid sequence of the first polypeptide chain is as shown in any one of SEQ ID NO: 25-27 or has at least 80% or at least 90% sequence identity with it, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 24 or has at least 80% or at least 90% sequence identity with it.

In some specific embodiments, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 28 or 29 or has at least 80% or at least 90% sequence identity with it, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 24 or has at least 80% or at least 90% sequence identity with it.

In some embodiments, a CLDN18.2/4-1BB binding protein is provided, comprising first and second polypeptide chains selected from the following:

(1) The amino acid sequence of the first polypeptide chain is shown in any one of SEQ ID NO: 25-27, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 24;

(2) The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO: 28 or 29, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO: 24.

In some embodiments, the CLDN18.2/4-1BB binding protein of this disclosure has an activity selected from at least one of the following:

(a) Binding to human 4-1BB or its epitopes with a _KD value ^≤10⁻⁷ ;

(b) When not cross-linked with CLDN18.2 (or not bound to CLDN18.2), the 4-1BB signaling pathway is weakly activated or not activated. For example, at a 100n antibody concentration, the activation level when not cross-linked with CLDN18.2 (or not bound to CLDN18.2) does not exceed 10% of the activity at the saturated antibody concentration when cross-linked with CLDN18.2 (or bound to CLDN18.2).

(c) When cross-linked with CLDN18.2 (or not bound to CLDN18.2), it strongly activates or strongly activates the 4-1BB signaling pathway, for example, EC ₅₀ is less than 1 nM;

(d) Activate T cells and/or promote T cell proliferation;

(e) Inhibits tumor growth;

(f) It has enhanced ADCC and/or ADCP capabilities.

For example, the activation detection of the 4-1BB signaling pathway in (b) and (c) refers to the 4-1BB/NF-κB luciferase reporter gene detection method in Example 2.

In some embodiments, the CLDN18.2/4-1BB binding protein of this disclosure is able to inhibit tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

In some embodiments, the CLDN18.2/4-1BB binding protein of this disclosure includes variants that have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the first or second polypeptide chain in any combination of (1)-(4) above; said amino acid mutations may be conservative substitutions, replacements or modifications, and/or deletions or additions that do not affect function.

In some embodiments, the CLDN18.2/4-1BB binding protein of this disclosure binds to or competitively binds to CLDN18.2 and/or 4-1BB, or binds to or competitively binds to the same epitopes of CLDN18.2 and/or 4-1BB.

In some embodiments, the CLDN18.2/4-1BB binding protein of this disclosure blocks the binding of the aforementioned CLDN18.2/4-1BB binding protein to CLDN18.2 and/or 4-1BB.

In some embodiments, a protein or molecule is provided that comprises any of the CLDN18.2/4-1BB binding proteins disclosed herein. For example, the protein or molecule is a conjugate, which may, for example, contain any detectable tag.

In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.0-8.0, for example, 4.5-7.5, 4.5-7.2, 4.5-7.0, 4.5-6.8, 4.5-6.5, 4.5-6.3, 4.5-6.0, 4.8-7.5, 4.8-7.2, 4.8-7.0, 4.8-6.8, 4.8-6.5, 4.8-6.3, 4.8 -6.0, 5.0-7.2, 5.0-7.1, 5.0-7, 5.0-6.9, 5.0-6.8, 5.0-6.7, 5.0-6.6, 5.0-6.5, 5.0-6.4, 5.0-6.3, 5.0-6.2, 5.0-6.1, 5.0-6.0, 5.3-7.0, 5.3-6.9, 5.3-6.8, 5.3-6.7, 5 5.3-6.6, 5.3-6.5, 5.3-6.4, 5.3-6.3, 5.3-6.2, 5.3-6.1, 5.3-6.0, 5.5-7.0, 5.5-6.9, 5.5-6.8, 5.5-6.7, 5.5-6.6, 5.5-6.5, 5.5-6.4, 5.5-6.3, 5.5-6.2, 5.5-6.1, 5.5-6 0, 5.6-7.0, 5.6-6.9, 5.6-6.8, 5.6-6.7, 5.6-6.6, 5.6-6.5, 5.6-6.4, 5.6-6.3, 5.6-6.2, 5.6-6.1, 5.5-6.0, 6.0-7.0, 6.0-6.9, 6.0-6.8, 6.0-6.7, 6.0-6.6 or 6.0-6.5.

In some embodiments, the buffer in the pharmaceutical composition has a pH of 4.5-7.5. In some embodiments, the buffer in the pharmaceutical composition has a pH of 4.8-7.2. In some embodiments, the buffer in the pharmaceutical composition has a pH of 5.0-7.0. In some embodiments, the buffer has a pH of 5.3-6.7. In some embodiments, the buffer has a pH of 5.5-7.0. In some embodiments, the buffer has a pH of 6.0-7.0. In some embodiments, the buffer in the pharmaceutical composition has a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6.

In some embodiments, the pH of the pharmaceutical composition differs from the pH of the buffer it contains by no more than ±1, for example, no more than ±0.5.

In some embodiments, the pH of the pharmaceutical composition is 4.0-8.0, for example 4.5-7.5, 4.5-7.2, 4.5-7.0, 4.5-6.8, 4.5-6.5, 4.5-6.3, 4.5-6.0, 4.8-7.5, 4.8-7.2, 4.8-7.0, 4.8-6.8, 4.8-6.5, 4.8-6.3, 4.8-6. 0, 5.0-7.2, 5.0-7.1, 5.0-7, 5.0-6.9, 5.0-6.8, 5.0-6.7, 5.0-6.6, 5.0-6.5, 5.0-6.4, 5.0-6.3, 5.0-6.2, 5.0-6.1, 5.0-6.0, 5.3-7.0, 5.3-6.9, 5.3-6.8, 5.3-6.7, 5.3 -6.6, 5.3-6.5, 5.3-6.4, 5.3-6.3, 5.3-6.2, 5.3-6.1, 5.3-6.0, 5.5-7.0, 5.5-6.9, 5.5-6.8, 5.5-6.7, 5.5-6.6, 5.5-6.5, 5.5-6.4, 5.5-6.3, 5.5-6.2, 5.5-6.1, 5.5-6. 0, 5.6-7.0, 5.6-6.9, 5.6-6.8, 5.6-6.7, 5.6-6.6, 5.6-6.5, 5.6-6.4, 5.6-6.3, 5.6-6.2, 5.6-6.1, 5.5-6.0, 6.0-7.0, 6.0-6.9, 6.0-6.8, 6.0-6.7, 6.0-6.6, or 6.0-6.5. In some embodiments, the pH of the pharmaceutical composition is 4.5-7.5. In some embodiments, the pH of the pharmaceutical composition is 4.8-7.2. In some embodiments, the pH of the pharmaceutical composition is 5.0-7.0. In some embodiments, the pH of the pharmaceutical composition is 5.3-6.7. In some embodiments, the pH of the buffer is 5.5-7.0. In some embodiments, the pH of the buffer is 6.0-7.0. In some embodiments, the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the pH of the pharmaceutical composition is about 5.6, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In some embodiments, the pH of the pharmaceutical composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6.

In some embodiments, the buffer concentration in the pharmaceutical composition is 0.1-60 mM, for example, 0.1-50 mM, 0.1-40 mM, 0.1-30 mM, 0.1-20 mM, 0.1-10 mM, 0.5-40 mM, 0.5-30 mM, 0.5-20 mM, 0.5-10 mM, 1-30 mM, 1-20 mM, 1-10 mM, 5-20 mM, or 5-10 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is 0.1-50 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is 0.5-40 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is 1-30 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is 5-20 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In some embodiments, the buffer concentration in the pharmaceutical composition is about 10 mM.

In some embodiments, the pharmaceutical composition further comprises one or more excipients selected from amino acids or their pharmaceutically acceptable salts, metal chelators, salts, and sugars.

In some embodiments, the amino acid is selected from one or more of methionine and proline.

In some embodiments, the sugar is selected from one or more of sucrose and trehalose. In some embodiments, the sugar is sucrose.

In some embodiments, the metal chelating agent is edetate or a pharmaceutically acceptable salt thereof. In some embodiments, the metal chelating agent is selected from monosodium edetate, disodium edetate, trisodium edetate, or tetrasodium edetate. In some embodiments, the metal chelating agent is disodium edetate.

In some embodiments, the salt is sodium chloride.

In some embodiments, the excipient is selected from one or more of sucrose, methionine or its pharmaceutically acceptable salt, proline or its pharmaceutically acceptable salt, edetidonic acid or its pharmaceutically acceptable salt, and sodium chloride.

In some embodiments, the excipient is selected from any one of the following: 1) sucrose; 2) sucrose and edetic acid or a pharmaceutically acceptable salt thereof, such as disodium edetate; 3) sucrose and methionine or a pharmaceutically acceptable salt thereof; 4) sucrose and sodium chloride; 5) proline or a pharmaceutically acceptable salt thereof. In some embodiments, the excipient is selected from: 1) sucrose; 2) sucrose and edetic acid or a pharmaceutically acceptable salt thereof, such as disodium edetate.

In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.01% w/v to 30% w/v, for example 0.01% w/v to 20% w/v, 0.01% w/v to 15% w/v, 0.01% w/v to 10% w/v, 0.01% w/v to 5% w/v, 0.01% w/v to 2.5% w/v, 0.05% w/v to 1%. 5% w/v, 0.05% w/v-10% w/v, 0.05% w/v-5% w/v, 0.05% w/v-2.5% w/v, 0.08% w/v-10% w/v, 0.08% w/v-5% w/v, 0.08% w/v-2.5% w/v, 0.1% w/v-5% w/v, or 0.1% w/v-2.5% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.01% w/v-20% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.05% w/v-15% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.08% w/v-10% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.08% w/v to 5% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is about 0.08% w/v, about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9% w/v, about 1% w/v, about 1.5% w/v, about 2% w/v, about 2.5% w/v, about 3% w/v, about 3.5% w/v, about 4% w/v, about 4.5% w/v, or about 5% w/v. In some embodiments, the concentration of the amino acid or its pharmaceutically acceptable salt is about 0.1% w/v or about 2.5% w/v.

In some embodiments, the concentration of the metal chelating agent is 0.0001% w/v to 1% w/v, for example 0.0001% w/v to 0.5% w/v, 0.0001% w/v to 0.1% w/v, 0.0001% w/v to 0.05% w/v, 0.0001% w/v to 0.01% w/v, 0.0001% w/v to 0.005% w/v, 0.0003% w/v to 0.1% w/v, 0.0003% w/v to 0. The concentrations are 0.0003% w/v to 0.01% w/v, 0.0003% w/v to 0.005% w/v, 0.0005% w/v to 0.05% w/v, 0.0005% w/v to 0.01% w/v, 0.0005% w/v to 0.005% w/v, 0.0008% w/v to 0.01% w/v, 0.0008% w/v to 0.005% w/v, or 0.001% w/v to 0.005% w/v. In some embodiments, the concentration of the metal chelating agent is 0.0001% w/v to 0.5% w/v. In some embodiments, the concentration of the metal chelating agent is 0.0003% w/v to 0.1% w/v. In some embodiments, the concentration of the metal chelating agent is 0.0005% w/v to 0.05% w/v. In some embodiments, the concentration of the metal chelating agent is 0.0008% w/v to 0.01% w/v. In some embodiments, the concentration of the metal chelating agent is about 0.0008% w/v, about 0.0009% w/v, about 0.001% w/v, about 0.002% w/v, about 0.003% w/v, about 0.004% w/v, about 0.005% w/v, about 0.006% w/v, about 0.007% w/v, about 0.008% w/v, about 0.009% w/v, or about 0.01% w/v. In some embodiments, the concentration of the metal chelating agent is about 0.002% w/v.

In some embodiments, the concentration of the salt is 0.001% w/v to 20% w/v, for example 0.005% w/v to 15% w/v, 0.005% w/v to 10% w/v, 0.005% w/v to 5% w/v, 0.005% w/v to 1% w/v, 0.005% w/v to 0.5% w/v, 0.01% w/v to 10% w/v. %w/v, 0.01%w/v-5%w/v, 0.01%w/v-1%w/v, 0.01%w/v-0.5%w/v, 0.05%w/v-5%w/v, 0.05%w/v-1%w/v, 0.05%w/v-0.5%w/v, 0.1%w/v-1%w/v, or 0.1%w/v-0.5%w/v. In some embodiments, the salt concentration is 0.005%w/v-15%w/v. In some embodiments, the salt concentration is 0.01%w/v-10%w/v. In some embodiments, the salt concentration is 0.05%w/v-5%w/v. In some embodiments, the salt concentration is 0.1%w/v-1%w/v. In some embodiments, the salt concentration is about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9% w/v, or about 1% w/v. In some embodiments, the salt concentration is about 0.5% w/v.

In some embodiments, the sugar concentration is 0.01% w/v to 40% w/v, for example 0.05% w/v to 35% w/v, 0.05% w/v to 30% w/v, 0.05% w/v to 25% w/v, 0.05% w/v to 20% w/v, 0.05% w/v to 15% w/v, 0.05% w/v to 10% w/v, 0.05% w/v to 5% w/v, 0.1% w/v to 30% w/v, 0.1% w/v to 25% w/v, 0.1% w/v to 20 ... %w/v-15%w/v, 0.1%w/v-10%w/v, 0.1%w/v-5%w/v, 0.5%w/v-25%w/v, 0.5%w/v-20%w/v, 0.5%w/v-15%w/v, 0.5%w/v-10%w/v, 0.5%w/v-5%w/v, 1%w/v-20%w/v, 1%w/v-15%w/v, 1%w/v-10%w/v, 1%w/v-5%w/v, 5%w/v-15%w/v, or 5%w/v-10%w/v. In some embodiments, the sugar concentration is 0.05%w/v-30%w/v. In some embodiments, the sugar concentration is 0.1%w/v-20%w/v. In some embodiments, the sugar concentration is 0.5% w/v to 15% w/v. In some embodiments, the sugar concentration is 1% w/v to 10% w/v. In some embodiments, the sugar concentration is about 1% w/v, about 1.5% w/v, about 2% w/v, about 2.5% w/v, about 3% w/v, about 3.5% w/v, about 4% w/v, about 4.5% w/v, about 5% w/v, about 5.5% w/v, about 6% w/v, about 6.5% w/v, about 7% w/v, about 7.5% w/v, about 8% w/v, about 8.5% w/v, about 9% w/v, about 9.5% w/v, or about 10% w/v. In some embodiments, the sugar concentration is about 4.5% w/v or about 8% w/v.

In some embodiments, the excipients are selected from any of the following:

1-1) 0.05% w/v - 30% w/v sucrose;

1-2) 0.1% w/v - 20% w/v sucrose;

1-3) 0.5% w/v - 15% w/v sucrose;

1-4) 1% w/v - 10% w/v sucrose;

1-5) 4.5% w/v or about 8% w/v sucrose.

In some embodiments, the excipients are selected from any of the following:

2-1) 0.05% w/v-30% w/v sucrose and 0.0001% w/v-0.5% w/v edemaic acid or its pharmaceutically acceptable salts, such as disodium edemaic acid;

2-2) 0.1% w/v-20% w/v sucrose and 0.0003% w/v-0.1% w/v edetate or its pharmaceutically acceptable salts, such as disodium edetate;

2-3) 0.5% w/v-15% w/v sucrose and 0.0005% w/v-0.05% w/v edetate or its pharmaceutically acceptable salts, such as disodium edetate;

2-4) 1% w/v-10% w/v sucrose and 0.0008% w/v-0.01% w/v edemaic acid or its pharmaceutically acceptable salts, such as disodium edemaic acid;

2-5) Approximately 8% w/v sucrose and approximately 0.002% w/v edetate or its pharmaceutically acceptable salt, such as disodium edetate.

In some embodiments, the excipients are selected from any of the following:

3-1) 0.05% w/v - 30% w/v sucrose and 0.01% w/v - 20% w/v methionine or its pharmaceutically acceptable salts;

3-2) 0.1% w/v-20% w/v sucrose and 0.05% w/v-15% w/v methionine or its pharmaceutically acceptable salts;

3-3) 0.5% w/v-15% w/v sucrose and 0.08% w/v-10% w/v methionine or its pharmaceutically acceptable salts;

3-4) 1% w/v-10% w/v sucrose and 0.08% w/v-5% w/v methionine or its pharmaceutically acceptable salts;

3-5) Approximately 8% w/v sucrose and approximately 0.1% w/v methionine or its pharmaceutically acceptable salt.

In some embodiments, the excipients are selected from any of the following:

4-1) 0.05% w/v - 30% w/v sucrose and 0.005% w/v - 15% w/v sodium chloride;

4-2) 0.1% w/v - 20% w/v sucrose and 0.01% w/v - 10% w/v sodium chloride;

4-3) 0.5% w/v - 15% w/v sucrose and 0.05% w/v - 5% w/v sodium chloride;

4-4) 1% w/v - 10% w/v sucrose and 0.1% w/v - 1% w/v sodium chloride;

4-5) Approximately 4.5% w/v sucrose and approximately 0.5% w/v sodium chloride.

In some embodiments, the excipients are selected from any of the following:

5-1) 0.01% w/v - 20% w/v proline or its pharmaceutically acceptable salts;

5-2) 0.05% w/v - 15% w/v proline or its pharmaceutically acceptable salts

5-3) 0.08% w/v - 10% w/v proline or its pharmaceutically acceptable salts;

5-4) 0.08% w/v - 5% w/v proline or its pharmaceutically acceptable salts;

5-5) Approximately 2.5% w/v proline or its pharmaceutically acceptable salt.

In some embodiments, the pharmaceutical compositions disclosed herein further comprise a surfactant. In some embodiments, the surfactant is an ionic or nonionic surfactant. In some embodiments, the surfactant is selected from polysorbate, polyhydroxyalkanoates, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroyl The surfactant is one or more of the following: aminopropyl betaine, cocarbamopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitoamide propyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitoamide propyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is polysorbate 80.

In some embodiments, the surfactant concentration in the pharmaceutical composition is 0.001% w/v to 1% w/v, for example, 0.005% w/v to 1% w/v, 0.005% w/v to 0.8% w/v, 0.005% w/v to 0.6% w/v, 0.005% w/v to 0.4% w/v, 0.005% w/v to 0.2% w/v, 0... .005%w/v-0.1%w/v, 0.005%w/v-0.09%w/v, 0.005%w/v-0.08%w/v, 0.005%w/v-0. 07% w/v, 0.005% w/v-0.06% w/v, 0.005% w/v-0.05% w/v, 0.008% w/v-0.5% w/v, 0.008 %w/v-0.4%w/v, 0.008%w/v-0.3%w/v, 0.008%w/v-0.2%w/v, 0.008%w/v-0.1%w/v, 0.008%w/v-0.09%w/v、0.008%w/v-0.08%w/v、0.008%w/v-0.07%w/v、0.008%w/v- 0.06% w/v, 0.008% w/v-0.05% w/v, 0.01% w/v-0.1% w/v, 0.01% w/v-0.09% w/v, 0.01% w/v-0.08% w/v, 0.01% w/v-0.07% w/v, 0.01% w/v-0.06% w/v, 0.01% w/v-0.05% w/v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.005% w/v-1% w/v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.008% w/v-0.2% w/v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.01% w/v-0.1% w/v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.001% w/v, about 0.002% w/v, about 0.003% w/v, about 0.004% w/v, about 0.005% w/v, about 0.006% w/v, about 0.007% w/v, about 0.008% w/v, about 0.009% w/v, about 0.01% w/v, about 0.02% w/v, about 0.03% w/v, about 0.04% w/v, about 0.05% w/v, about 0.06% w/v, about 0.07% w/v, about 0.08% w/v, about 0.09% w/v, or about 0.1% w/v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.02% w/v, about 0.04% w/v, or about 0.06% w/v.

In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein in the pharmaceutical composition is 0.01-500 mg/mL, for example 0.05-400 mg/mL, 0.05-350 mg/mL, 0.05-300 mg/mL, 0.05-250 mg/mL, 0.05-200 mg/mL, 0.05-150 mg/mL, 0.05-100 mg/mL, 0.05-90 mg/mL, 0.05-80 mg/mL, 0.05-70 mg/mL, 0.05-60 mg/mL, 0.05-50 mg/mL, 0.05-45 mg/mL, 0.05-40 mg/mL, 0.05 -30mg/mL, 0.1-300mg/mL, 0.1-250mg/mL, 0.1-200mg/mL, 0.1-150mg/mL, 0.1-100mg/mL, 0.1-90mg/mL, 0.1-80mg/mL, 0.1-70mg/mL, 0.1-60mg/mL, 0. 1-50mg/mL, 0.1-45mg/mL, 0.1-40mg/mL, 0.1-30mg/mL, 0.5-200mg/mL, 0.5-150mg/mL, 0.5-100mg/mL, 0.5-90mg/mL, 0.5-80mg/mL, 0.5-70mg/mL, 0.5- 60mg/mL, 0.5-50mg/mL, 0.5-45mg/mL, 0.5-40mg/mL, 0.5-30mg/mL, 1-100mg/mL, 1-90mg/mL, 1-80mg/mL, 1-70mg/mL, 1-60mg/mL, 1-50mg/mL, 1-45mg/ mL, 1-40mg/mL, 1-30mg/mL, 5-80mg/mL, 5-70mg/mL, 5-60mg/mL, 5-50mg/mL, 5-40mg/mL, 5-30mg/mL, 10-60mg/mL, 10-50mg/mL, 10-40mg/mL, 10-30mg/m L, 50-500 mg/mL, 50-400 mg/mL, 50-350 mg/mL, 50-300 mg/mL, 50-250 mg/mL, 50-200 mg/mL, 50-150 mg/mL, 50-100 mg/mL, 50-90 mg/mL, 50-80 mg/mL, 20-500 mg/mL, 20-400 mg/mL, 20-350 mg/mL, 20-300 mg/mL, 20-250 mg/mL, 20-200 mg/mL, 20-150 mg/mL, 20-100 mg/mL, 20-90 mg/mL, 20-80 mg/mL, or 20-50 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 0.05-300 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 0.1-200 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 0.5-100 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 1-80 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 1-50 mg/mL, 1-45 mg/mL, or 1-30 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is 10-30 mg/mL or 10-50 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is about 1 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, or about 80 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, or about 50 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is ≥20 mg/mL, for example, 20-200 mg/mL, 20-100 mg/mL, or 20-50 mg/mL. In some embodiments, the concentration of the CLDN18.2/4-1BB binding protein is <50 mg/mL, for example, 1-45 mg/mL, 1-40 mg/mL, or 1-30 mg/mL.

In some embodiments, this disclosure provides a pharmaceutical composition comprising a CLDN18.2/4-1BB binding protein (e.g., 1903x C5_V2-YTI-AA with heavy and light chain sequences of SEQ ID NO: 27 and 24), comprising or comprising any group thereof:

A) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Polysorbates, such as polysorbate 80;

sucrose;

B) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Polysorbates, such as polysorbate 80;

Sucrose and edetate or their medicinal salts, such as sucrose and disodium edetate;

C) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Polysorbates, such as polysorbate 80;

Sucrose and methionine or their medicinal salts;

D) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Polysorbates, such as polysorbate 80;

Sucrose and sodium chloride;

E) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Polysorbates, such as polysorbate 80;

Proline or its medicinal salts;

F) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

G) Any of the aforementioned CLDN18.2/4-1BB binding proteins;

Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

Sucrose and/or edetate or its pharmaceutically acceptable salts, such as sucrose and/or disodium edetate;

H) Any one of A)-G) above, wherein the buffer has a pH of 4.5-7.5, for example, a pH of 4.8-7.2, 5.0-7.0 or 5.3-6.7;

I) Any of A)-H) above, the pharmaceutical composition further comprises water for injection;

J) In any of A)-I) above, the pH of the pharmaceutical composition is 4.5-7.5, for example, pH is 4.8-7.2, 5.0-7.0 or 5.3-6.7;

K) Any one of A)-J) above, wherein the pharmaceutical composition is a liquid formulation;

L) Any one of A)-K) above, wherein the pharmaceutical composition is a lyophilized formulation.

In some embodiments, this disclosure provides a pharmaceutical composition comprising a CLDN18.2/4-1BB binding protein (e.g., 1903x C5_V2-YTI-AA with heavy and light chain sequences of SEQ ID NO: 27 and 24), comprising or comprising any group thereof:

A-1)

0.05-300 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.1-50mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.001% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.05% w/v - 30% w/v sucrose;

The pH of the composition is 4.5-7.5;

A-2)

0.1-200 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.5-40mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.005% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.1% w/v - 20% w/v sucrose;

The pH of the composition is 4.8-7.2;

A-3)

0.5-100 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

1-30mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.008% w/v - 0.2% w/v polysorbate, for example, polysorbate 80;

0.5% w/v - 15% w/v sucrose;

The pH of the composition is 5.0-7.0;

A-4)

1-80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

5-20mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

1% w/v - 10% w/v sucrose;

The pH of the composition is 5.3-6.7;

B-1)

0.05-300 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.1-50mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.001% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.05% w/v-30% w/v sucrose and 0.0001% w/v-0.5% w/v edetate or its pharmaceutically acceptable salts, such as 0.05% w/v-30% w/v sucrose and 0.0001% w/v-0.5% w/v disodium edetate;

The pH of the composition is 4.5-7.5;

B-2)

0.1-200 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.5-40mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.005% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.1% w/v-20% w/v sucrose and 0.0003% w/v-0.1% w/v edemaic acid or its pharmaceutically acceptable salts, such as 0.1% w/v-20% w/v sucrose and 0.0003% w/v-0.1% w/v disodium edemaic acid;

The pH of the composition is 4.8-7.2;

B-3)

0.5-100 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

1-30mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.008% w/v - 0.2% w/v polysorbate, for example, polysorbate 80;

0.5% w/v-15% w/v sucrose and 0.0005% w/v-0.05% w/v edetate or its pharmaceutically acceptable salts, such as 0.5% w/v-15% w/v sucrose and 0.0005% w/v-0.05% w/v disodium edetate;

The pH of the composition is 5.0-7.0;

B-4)

1-80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

5-20mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

1% w/v-10% w/v sucrose and 0.0008% w/v-0.01% w/v edemaic acid or its pharmaceutically acceptable salts, such as 1% w/v-10% w/v sucrose and 0.0008% w/v-0.01% w/v disodium edemaic acid;

The pH of the composition is 5.3-6.7;

The pharmaceutical composition further comprises water for injection, according to any one of A-1 to A-4 and B-1 to B-4 above.

Any of A-1 to A-4) and B-1 to B-4) above, wherein the pharmaceutical composition is a lyophilized formulation, for example, the concentration of the CLDN18.2/4-1BB binding protein is 20-50 mg/ml;

The pharmaceutical composition is any one of B-1 to B-4 above, wherein the concentration of the CLDN18.2/4-1BB binding protein is <50 mg/mL, for example 1-45 mg/mL, 1-40 mg/mL or 1-30 mg/mL, and the pharmaceutical composition is a liquid formulation.

In some embodiments, this disclosure provides a pharmaceutical composition comprising a CLDN18.2/4-1BB binding protein (e.g., 1903x C5_V2-YTI-AA with heavy and light chain sequences of SEQ ID NO: 27 and 24), comprising or comprising any group thereof:

C-1)

0.05-300 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.1-50mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.001% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.05% w/v - 30% w/v sucrose and 0.01% w/v - 20% w/v methionine or its pharmaceutically acceptable salts;

The pH of the composition is 4.5-7.5;

C-2)

0.1-200 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.5-40mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.005% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.1% w/v-20% w/v sucrose and 0.05% w/v-15% w/v methionine or its pharmaceutically acceptable salts;

C-3)

0.5-100 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

The pH of the composition is 4.8-7.2;

1-30mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.008% w/v - 0.2% w/v polysorbate, for example, polysorbate 80;

0.5% w/v-15% w/v sucrose and 0.08% w/v-10% w/v methionine or its pharmaceutically acceptable salts;

The pH of the composition is 5.0-7.0;

C-4)

1-80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

5-20mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

1% w/v-10% w/v sucrose and 0.08% w/v-5% w/v methionine or its pharmaceutically acceptable salts;

The pH of the composition is 5.3-6.7;

D-1)

0.05-300 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.1-50mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.001% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.05% w/v - 30% w/v sucrose and 0.005% w/v - 15% w/v sodium chloride;

The pH of the composition is 4.5-7.5;

D-2)

0.1-200 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.5-40mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.005% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.1% w/v-20% w/v sucrose and 0.01% w/v-10% w/v sodium chloride;

D-3)

0.5-100 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

1-30mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.008% w/v - 0.2% w/v polysorbate, for example, polysorbate 80;

0.5% w/v - 15% w/v sucrose and 0.05% w/v - 5% w/v sodium chloride;

The pH of the composition is 5.0-7.0;

D-4)

1-80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

5-20mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

1% w/v-10% w/v sucrose and 0.1% w/v-1% w/v sodium chloride;

The pH of the composition is 5.3-6.7;

E-1)

0.05-300 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.1-50mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.001% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.01% w/v - 20% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is 4.5-7.5;

E-2)

0.1-200 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

0.5-40mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.005% w/v - 1% w/v polysorbate, for example, polysorbate 80;

0.05% w/v - 15% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is 4.8-7.2;

E-3)

0.5-100 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

1-30mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.008% w/v - 0.2% w/v polysorbate, for example, polysorbate 80;

0.08% w/v - 10% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is 5.0-7.0;

E-4)

1-80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

5-20mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

0.08% w/v - 5% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is 5.3-6.7;

The pharmaceutical composition further comprises water for injection, and includes any one of C-1 to C-4), D-1 to D-4), and E-1 to E-4) above.

In some embodiments, this disclosure provides a pharmaceutical composition comprising a CLDN18.2/4-1BB binding protein (e.g., 1903x C5_V2-YTI-AA with heavy and light chain sequences of SEQ ID NO: 27 and 24), comprising or comprising any group thereof:

A-5)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate 80;

Approximately 8% w/v sucrose;

The pH of the composition is approximately 6.0;

A-6)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.02% w/v polysorbate 80;

Approximately 8% w/v sucrose;

The pH of the composition is approximately 6.0;

A-7)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.04% w/v polysorbate 80;

Approximately 8% w/v sucrose;

The pH of the composition is approximately 6.0;

A-8)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.06% w/v polysorbate 80;

Approximately 8% w/v sucrose;

The pH of the composition is approximately 6.0;

A-9)

10 mg/mL-50 mg/mL or 10 mg/mL-30 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v or 0.02% w/v - 0.06% w/v polysorbate 80;

Approximately 8% w/v sucrose;

The pH of the composition is approximately 6.0;

B-5)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate 80;

About 8% w/v sucrose and about 0.002% w/v edetate or its pharmaceutically acceptable salt, such as about 8% w/v sucrose and about 0.002% w/v disodium edetate;

The pH of the composition is approximately 6.0;

B-6)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.02% w/v polysorbate 80;

About 8% w/v sucrose and about 0.002% w/v edetate or a pharmaceutically acceptable salt thereof, such as about 8% w/v sucrose and about 0.002% w/v disodium edetate or a pharmaceutically acceptable salt thereof;

The pH of the composition is approximately 6.0;

B-7)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.04% w/v polysorbate 80;

About 8% w/v sucrose and about 0.002% w/v edetate or a pharmaceutically acceptable salt thereof, such as about 8% w/v sucrose and about 0.002% w/v disodium edetate or a pharmaceutically acceptable salt thereof;

The pH of the composition is approximately 6.0;

B-8)

Approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

Approximately 0.06% w/v polysorbate 80;

About 8% w/v sucrose and about 0.002% w/v edetate or a pharmaceutically acceptable salt thereof, such as about 8% w/v sucrose and about 0.002% w/v disodium edetate or a pharmaceutically acceptable salt thereof;

The pH of the composition is approximately 6.0;

B-9)

10 mg/mL - 50 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins, for example, 10 mg/mL - 30 mg/mL;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v or 0.02% w/v - 0.06% w/v polysorbate 80;

About 8% w/v sucrose and about 0.002% w/v edetate or a pharmaceutically acceptable salt thereof, such as about 8% w/v sucrose and about 0.002% w/v disodium edetate or a pharmaceutically acceptable salt thereof;

The pH of the composition is approximately 6.0;

The pharmaceutical composition of any one of A-5 to A-9) and B-5 to B-9) above, wherein the pH is 5.5-6.5, for example about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4 or about 6.5.

The pharmaceutical composition of any one of A-5 to A-9) and B-5 to B-9) above further comprises water for injection;

Any of A-5 to A-9) and B-5 to B-9) above, wherein the pharmaceutical composition is a lyophilized formulation, for example, the concentration of the CLDN18.2/4-1BB binding protein is 20-50 mg/ml;

The pharmaceutical composition comprises any one of B-5 to B-9 above, wherein the concentration of the CLDN18.2/4-1BB binding protein is <50 mg/mL, for example 1-45 mg/mL, 1-40 mg/mL, or 1-30 mg/mL, and the pharmaceutical composition is a liquid formulation.

In some embodiments, this disclosure provides a pharmaceutical composition comprising a CLDN18.2/4-1BB binding protein (e.g., 1903x C5_V2-YTI-AA with heavy and light chain sequences of SEQ ID NO: 27 and 24), comprising or comprising any group thereof:

C-5) approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

Approximately 8% w/v sucrose and approximately 0.1% w/v methionine or its pharmaceutically acceptable salts;

The pH of the composition is approximately 6.0;

C-6) 10 mg/mL-50 mg/mL or 10 mg/mL-30 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v or 0.02% w/v - 0.06% w/v polysorbate 80;

Approximately 8% w/v sucrose and approximately 0.1% w/v methionine or its pharmaceutically acceptable salts;

The pH of the composition is approximately 6.0;

D-5) approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

Approximately 4.5% w/v sucrose and approximately 0.5% w/v sodium chloride;

The pH of the composition is approximately 6.0;

D-6) 10 mg/mL-50 mg/mL or 10 mg/mL-30 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v or 0.02% w/v - 0.06% w/v polysorbate 80;

Approximately 4.5% w/v sucrose and approximately 0.5% w/v sodium chloride;

The pH of the composition is approximately 6.0;

E-5) approximately 10 mg/mL, approximately 20 mg/mL, approximately 30 mg/mL, approximately 40 mg/mL, approximately 50 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL or approximately 80 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v polysorbate, such as polysorbate 80;

Approximately 2.5% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is approximately 6.0;

E-6) 10 mg/mL-50 mg/mL or 10 mg/mL-30 mg/mL of any of the aforementioned CLDN18.2/4-1BB binding proteins;

Approximately 10 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

0.01% w/v - 0.1% w/v or 0.02% w/v - 0.06% w/v polysorbate 80;

Approximately 2.5% w/v proline or its pharmaceutically acceptable salts;

The pH of the composition is approximately 6.0;

The pharmaceutical composition of any one of C-5 to C-6), D-5 to D-6), and E-5 to E-6 above, wherein the concentration of polysorbate 80 is about 0.02% w/v, about 0.04% w/v, or about 0.06% w/v;

The pharmaceutical composition of any one of C-5 to C-6), D-5 to D-6), and E-5 to E-6 above further comprises water for injection.

This disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of mixing CLDN18.2/4-1BB binding protein with a buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer (e.g., histidine-histidine hydrochloride).

In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is a liquid formulation, such as an injectable formulation. In some embodiments, the solvent for the liquid formulation is water, saline, or a glucose solution.

This disclosure also provides a lyophilized formulation, characterized in that the lyophilized formulation, after reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

This disclosure also provides a lyophilized formulation obtained by freeze-drying a pharmaceutical composition as described in any of the preceding embodiments. In some embodiments, the freeze-drying process sequentially includes pre-freezing, primary drying, and secondary drying steps.

This disclosure provides a reconstituted solution prepared by reconstituted the aforementioned lyophilized formulation. In some embodiments, the reconstituted solution is selected from, but is not limited to, water for injection, physiological saline, or glucose solution.

This disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as described above, a lyophilized formulation as described above, or a reconstituted solution as described above. In some embodiments, the container is a neutral borosilicate glass vial for injection. In some embodiments, the article of manufacture includes a pharmaceutical instruction leaflet.

This disclosure provides the use of the aforementioned pharmaceutical compositions, pharmaceutical compositions prepared by the aforementioned methods, the aforementioned lyophilized formulations, or the aforementioned reconstituted solutions as medicines for improving, alleviating, treating, or preventing diseases.

In some embodiments, the aforementioned disease is a proliferative disorder or any other disease or condition characterized by uncontrolled cell growth, such as cancer. In this disclosure, cancer and tumor are used interchangeably.

In some implementations, the aforementioned cancer is a solid tumor or a hematologic malignancy.

In some implementations, the aforementioned cancer is advanced or metastatic.

In some embodiments, the aforementioned disease is CLDN18.2-related or CLDN18.2-positive, such as CLDN18.2-positive cancer. In some embodiments, diseases related to Claudin18.2 or CLDN18.2-positive can be diagnosed by detecting or measuring cells expressing Claudin18.2 using antibodies or antibody fragments of this disclosure. To detect cells expressing the polypeptide, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, immunohistostaining, etc., being preferred. Additionally, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) system, etc., can be used. In this disclosure, there are no particular limitations on the test sample used to detect or measure the target antigen (e.g., Claudin18.2), as long as it has the potential to contain cells expressing the target antigen (e.g., Claudin18.2), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

In some implementations, the aforementioned cancers are selected from the following or combinations thereof: lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, or any other disease or condition characterized by uncontrolled cell growth.

Terminology Definition

To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as having an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.”

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

The CLDN18 protein, or CLDN18, is a protein encoded by the Claudin18 gene in humans and belongs to the tight junction protein family. Claudin-18 controls molecular flow between cellular layers. The Claudin-18 protein structure includes four transmembrane regions, two extracellular loops, and its N-terminus and C-terminus are located in the cytoplasm. Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, which differ from each other only by eight amino acids in the first extracellular loop. The expression distribution of Claudin 18.1 and Claudin 18.2 differs; Claudin 18.1 is selectively expressed in normal lung cells, while Claudin 18.2 expression is highly restricted in normal cells but is frequently ectopically activated and overexpressed in various tumors (such as gastric cancer, lung cancer, and pancreatic cancer).

CD16, also known as FcγRIII, is a receptor with low affinity for the Fc fragment of IgGs associated with antibody-dependent cytotoxicity (ADCC). Human FcγRIII has two subtypes, FcγRIIIA (i.e., CD16A) and FcγRIIIB (i.e., CD16B), which share 96% sequence identity in their extracellular immunoglobulin-binding regions (van de Winkel and Capel, 1993, Immunol Today 14(5): 215-221). CD16A is a transmembrane receptor expressed on macrophages, mast cells, and NK cells. On NK cells, the α-chain of CD16A binds to the immune receptor tyrosine activation motif (ITAM) containing the FcεRIγ-chain and/or the T-cell receptor (TCR)/CD3ζ-chain, inducing signal transduction and leading to cytokine production and cytotoxicity. CD16B, as a glycosyl-phosphatidylinositol (GPI)-anchored receptor (FcγRIIIB isoform), is present on polymorphonuclear leukocytes (PMNs), but this receptor does not induce tumor cell killing (van de Winkel and Capel, 1993, supra). Furthermore, CD16B exists as a soluble receptor in serum, and once it binds to antibodies in vivo, it may cause side effects by forming immune complexes.

"4-1BB protein" or "4-1BB", also known as CD137 or tumor necrosis factor receptor superfamily 9, is a member of the TNF receptor superfamily (TNFRSF) and a co-stimulatory molecule expressed on the surface of CD8+ and CD4+ T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells, and neutrophils. 4-1BB is a co-stimulatory molecule expressed after immune cells are activated. Human 4-1BB protein is registered in NCBI under accession number NP_001552.2. In this disclosure, "4-1BB" may optionally include any such protein or fragments or variants thereof, including (but not limited to) known or wild-type 4-1BB as described in this disclosure, and any naturally occurring splice variants, amino acid variants, or isoforms, such as human 4-1BB as shown in SEQ ID NO: 11.

The term "4-1BB binding protein" encompasses any molecule capable of specifically binding to 4-1BB or its epitopes, including but not limited to antibodies against 4-1BB as defined herein, their antigen-binding fragments, or conjugates thereof. The "4-1BB binding protein" of this disclosure may contain at least one (e.g., 1, 2, 3, 4, 5, 6, or more) immunoglobulin single variable domain (e.g., VHH) that binds to 4-1BB. In addition to containing the immunoglobulin single variable domain of 4-1BB, the "4-1BB binding protein" of this disclosure may also contain a linker and/or a portion having effector function, such as a half-life extension portion (e.g., an immunoglobulin single variable domain binding to serum albumin) and/or a fusion partner (e.g., serum albumin) and/or a conjugated polymer (e.g., PEG) and/or an Fc region.

"CLDN18.2/4-1BB binding protein" encompasses any molecule capable of specifically binding to CLDN18.2 protein or its epitope and 4-1BB protein or its epitope, including but not limited to antibodies, peptides, fusion proteins of antibodies and peptides, or conjugates thereof. In some embodiments, "CLDN18.2/4-1BB binding protein" encompasses the anti-CLDN18.2/4-1BB bispecific antibody in the embodiments of this disclosure.

The term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies disclosed herein can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable in several embodiments. Generally, the antibodies disclosed herein are recombinant antibodies. As used herein, “recombinant” refers to products such as cells or nucleic acids, proteins, or vectors, indicating that said cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that said cells are derived from such modified cells. For example, recombinant cells express genes not present in native (non-recombinant) cell forms or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

"Antigen-binding fragments" encompass single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or quadrivalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

The determination or definition of CDRs can be accomplished by resolving the structure of the antibody and/or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues at the antibody binding site. This can be achieved using any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformation definition. The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a suite of computer programs produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened depending on the prediction or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. Correspondences between various numbering systems are well known to those skilled in the art and are exemplary, as shown in Table 1 below.

Table 1. Relationship between CDR numbering systems

The CDR amino acid residues in the VL and VH regions of the disclosed antibody conform to the known Kabat numbering system in terms of both number and position.

"Effector function" in this disclosure refers to biological activities attributable to the Fc region of an antibody and varying with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), Fc receptor (FcR) binding, cytokine secretion, antigen uptake by antigen-presenting cells mediated by immune complexes, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. For example, ADCC is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells that specifically bind to antibodies or their derivatives containing the Fc region. "Enhanced ADCC" is defined as an increase in the number of target cells lysed within a given time period by means of the ADCC mechanism defined above, with a given concentration of antibody in the target cell periphery medium, and/or a decrease in the concentration of antibody in the target cell periphery medium required to achieve the lysis of a given number of target cells within a given time period by means of the ADCC mechanism. ADCC enhancement is relative to ADCC mediated by the same antibody generated from the same type of host cells but not engineered, using the same standard production, purification, formulation, and storage methods known to those skilled in the art. For example, enhancement in ADCC mediated by an antibody containing an amino acid substitution that enhances ADCC in its Fc region is relative to ADCC mediated by the same antibody that does not have this amino acid substitution in the Fc region. Suitable assays for measuring ADCC are known in the art (see, for example, WO2006/082515 or WO2012/130831).

A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for specific functional properties of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and/or the domain itself.

An "immunoglobulin domain" refers to a globular region of an antibody chain (such as the chain of a conventional tetrapeptide chain antibody or a heavy chain antibody), or a polypeptide that is essentially composed of such globular regions. The immunoglobulin domain is characterized by its ability to maintain the immunoglobulin folding characteristics of the antibody molecule.

An immunoglobulin variable domain refers to an immunoglobulin domain essentially composed of four "frame regions," referred to in the art and hereinafter as "frame region 1" or "FR1," "frame region 2" or "FR2," "frame region 3" or "FR3," and "frame region 4" or "FR4," respectively. These frame regions are separated by three "complementarity-determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

"Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the complementarity-determining region (CDR) of that variable domain.

"Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH/VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH, and/or camelified VH, such as camelified human VH), IgNARs, domains, (single-domain) antibodies (such as dAbs ^™ ) that are VH domains or derived from VH domains, and (single-domain) antibodies (such as dAbs ^™ ) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of a single variable domain of an immunoglobulin is the “VHH domain” (or simply “VHH”) as defined below.

"VHH," also known as heavy chain single-domain antibody, VHH, _VHH domain, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., "antibody lacking a light chain"). (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the "VH domain" or VH) and the light chain variable domain (referred herein as the "VL domain" or VL) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. Terms include "heavy chain single-domain antibody,""VHHdomain,""VHH,"" _VHH domain,""VHH antibody fragment," and "VHH antibody." as well as" The term "domain"("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) is used interchangeably. VHHs include, but are not limited to, naturally occurring antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. The total number of amino acid residues in a VHH will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94/04678.

As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

"Humanized antibody," also known as CDR-grafted antibody, refers to an antibody generated by grafting a non-human CDR sequence into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. To avoid a decrease in activity along with a decrease in immunogenicity, the variable region of the fully human antibody can be minimally reverse-mutated to maintain activity. Examples of "humanization" include the substitution of a VHH domain derived from camelids with one or more amino acid residues in the original VHH sequence by replacing one or more amino acid residues present at the corresponding position in the VH domain of a conventional human tetrapeptide chain antibody (also referred to herein as "sequence optimization"; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by providing one or more mutations that enhance the properties of the VHH, such as removing potential post-translational modification sites). The humanized VHH domain may contain one or more fully human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods include protein surface amino acid resurfacing and antibody humanization using a universal framework grafting (CDR) method, which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes, for example, can be found in the VBase human germline sequence database and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

"Affinity-matured" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs) compared to parental antibodies that do not possess such alterations, resulting in improved affinity of the antibody for the antigen. For example, "affinity-matured" 4-1BB binding proteins or anti-4-1BB antibodies have one or more alterations in one or more CDRs, resulting in increased affinity for the antigen compared to their parental antibodies. Affinity-matured antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10: 779-783; or Barbas et al., 1994, Proc. Nat. Acad. Sci., USA 91: 3809-3813; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol. 155: 1994-2004. Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996.

Typically, the 4-1BB binding protein and CLDN18.2/4-1BB binding protein of this disclosure will bind to the antigen or target protein (i.e., 4-1BB, CLDN18.2) with a dissociation constant ( _KD ) preferably ^10⁻⁷ to ^10⁻¹⁰ mol/L (M), more preferably ^10⁻⁸ to ^10⁻¹⁰ mol/L, even more preferably ^10⁻⁹ to ^10⁻¹⁰ or lower, as measured in Biacore, KinExA, or Fortibio assays, and/or with an association constant (KA) of at least ^10⁻⁷ M, preferably at least ^10⁻⁸ M, more preferably at least ^10⁻⁹ M, and more preferably at least ^10⁻¹⁰ M. Any _KD value greater than ^10⁻⁴ M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described in this disclosure.

An epitope is a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining the binding of an epitope to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

"Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or a portion thereof with an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant ( _KD ). _KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). The rate constants corresponding to the binding and dissociation of a monovalent complex are referred to as the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. _KD is related to ka and kd by the equation _KD = kd/ka. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures using methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, the _KD can be determined using a dual-filtration nitrocellulose filter combined with an assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for assessing the binding ability of antibodies against target antigens are known in the art, including, for example, ELISA, Western blotting, RIA and flow cytometry analyses, as well as other assays exemplified elsewhere in this disclosure. Antibody binding kinetics and binding affinity can also be evaluated using standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using a Biacore ^™ system or KinExA. Binding affinity associated with different molecular interactions can be compared by comparing the _KD values of individual antibody/antigen complexes, for example, a comparison of the binding affinity of different antibodies for a given antigen. Similarly, the specificity of an interaction can be evaluated by determining and comparing the _KD value of the target interaction (e.g., a specific interaction between an antibody and an antigen) with the _KD value of a non-target interaction (e.g., a control antibody known not to bind to 4-1BB, CLDN18.2).

"Conservative substitution" refers to the substitution with another amino acid residue that has properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

"Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same nucleotide or amino acid monomer—for example, if every position in two DNA molecules is occupied by the same nucleotide—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

The terms "nucleic acid" and "polynucleotide" are used interchangeably in this disclosure and refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

"Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and/or transformed in vivo with the polynucleotides of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

"Inhibit" or "block" are used interchangeably and cover both partial and complete inhibition/blockage. "Inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

"Inhibition of growth" or "growth inhibition" refers to suppressing the growth or proliferation of cells.

"Proliferative disease" refers to a condition associated with a certain degree of abnormal cell proliferation. In one implementation, proliferative disease refers to cancer. "Tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "proliferative disease," and "tumor" are not mutually exclusive when used in this disclosure.

"Cancer prevention" refers to delaying, suppressing, or preventing the onset of cancer in subjects whose cancer development or the initiation of tumor development has not been confirmed, but whose cancer susceptibility has been identified through methods such as genetic screening or other means. This also includes treating subjects with precancerous conditions to halt the progression of said precancerous conditions to malignancy or to cause their regression.

As used herein, the terms “about” or “approximately” mean a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above a standard deviation of 1. Alternatively, “about” or “substantially comprises” may mean a range of up to ±20%, for example, about 5.5 pH means pH 5.5 ± 1.1. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially comprises” should be assumed to be within an acceptable margin of error for that specific value.

"Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include tris(hydroxymethyl)aminomethane (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

"Citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, and magnesium citrate. A preferred citrate buffer is sodium citrate buffer.

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., with histidine-hydrochloride buffer or histidine-acetate buffer being preferred. Histidine-acetate buffer is prepared by histidine and acetic acid, and histidine-hydrochloride buffer is prepared by histidine and histidine hydrochloride, or by histidine and hydrochloric acid.

"Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine-histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. The preferred acetate buffer is sodium acetate.

"Pharmaceutical composition" refers to a mixture containing one or more antibodies described herein, along with other chemical components, such as physiological/pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to the organism, and enhance the absorption of the active ingredient to exert its biological activity.

In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

"Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step after the liquid or solution form has been processed.

"Giving," "applying," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean treatment, such as of cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

"Treatment" means administering, either internally or externally, a therapeutic agent, such as a pharmaceutical composition comprising any of the binding proteins of this disclosure or thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

"Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects in this disclosure may be animal or human subjects.

"Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And/or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and/or" as used in phrases such as "A and/or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to."

In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans.

The pharmaceutical compositions described in this disclosure achieve a stable effect: the CLDN18.2/4-1BB binding protein substantially retains its physical and/or chemical stability and/or biological activity after storage; for example, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

Stable pharmaceutical formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for 1 month, 3 months, or 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: degradation of antibody monomers typically not exceeding about 10%, for example, not exceeding about 5%, as determined by SEC-HPLC. Variations in the concentration, pH, and osmotic pressure of the formulation not exceeding ±20%, for example, not exceeding ±10%. Formation of aggregates typically not exceeding about 10%, for example, not exceeding about 5%.

If, after visual inspection of color and/or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and/or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation.

If an antibody does not show significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the CLDN18.2/4-1BB binding protein "retains its biological activity" in the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

The equipment and methods used in the testing process are as follows:

Appearance:

Using the visual method, the sample vials were wiped clean, and the color, clarity, and visible foreign matter of the samples were observed under a light intensity of 1000–1500 lx on both a white background and a black background of a clarity meter.

Appearance inspection instrument: Jingtuo Instruments YB-2A clarity tester.

pH value:

The pH value was measured using a potentiometric method, taking 200 μL of sample and using a micro pH meter calibrated with standard solution.

pH measuring instrument: Mettler Toledo, S210.

SEC size exclusion chromatography:

An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, and A total is the sum of the peak areas of all peaks.)

Instruments used for SEC determination: Agilent 1260-Bio; Column: Waters, XBrige SEC (300×7.8mm 3.5μm)

NR-CE capillary gel electrophoresis:

An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

Non-reduced CE purity percentage = A_{main peak} / A_total * 100% (A_{main peak} is the peak area of the main peak in the sample, and A_total is the sum of the areas of all peaks).

CE testing instrument: Sciex PA800plus

icIEF imaging capillary isofocusing electrophoresis:

A technique for separating proteins based on their different isoelectric points (pI).

icIEF main peak content percentage = main peak area / total area * 100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

The instrument used for icIEF measurement is manufactured by Protein Simple, model Muarice.

Protein concentration determination:

Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop 2000, optical path length: 1 mm.

Exemplary antibody drug formulation preparation process

Step 1: The formulation stock solution prepared by mixing CLDN18.2/anti-4-1BB multifunctional antibody with the excipients in the following prescribed amounts is filtered through a 0.22μm filter cartridge for sterilization, and the filtrate is collected.

Step 2: Adjust the filling volume, use vials for filling, take samples at the beginning, middle and end of filling to test the difference in filling volume, and stopper the vials.

Step 3: Start the capping machine to cap the caps.

Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling or poor appearance. Print paper box labels, fold the paper boxes, pack them, and affix paper box labels.

Attached Figure Description

Figure 1 shows the FACS binding test results of the antibody to be tested with human 4-1BB antigen.

Figure 2 shows the results of 4-1BB/NF-κB luciferase reporter gene assay of the antibody before and after FcγRIIb crosslinking.

Figure 3 shows the activation effect of anti-4-1BB antibody C5 and its humanized antibodies C5_V1, C5_V2, and C5_V3 on the NF-κB signaling pathway, with Urelumab and isotype IgG1 antibody used as controls.

Figure 4 is a schematic diagram of the structure of the CLDN18.2/4-1BB bispecific antibody.

Figure 5 shows the activation detection results of the NF-κB signaling pathway by the modified C5_V2-YTI antibody 1903x C5_V2-YTI-LALA, with 1903x C5_V2-LALA, TJ-CD4B, and isotype IgG1 used as controls.

Figure 6 shows the CLDN18.2-dependent 4-1BB/NF-κB luciferase reporter gene assay results of CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2. The left side shows CLDN18.2 before cross-linking, and the right side shows CLDN18.2 after cross-linking. TJ-CD4B, ADG-106, and isotype IgG1 were used as controls in both cases.

Figures 7A and 7B show the activation detection results of T lymphocytes from donor 1 and donor 2 by the CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2-YTI, with TJ-CD4B and isotype IgG1 used as controls.

Figure 8 shows the results of NK cell killing of T cells induced by CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2-YTI, with isotype IgG1 as a control.

Figures 9A and 9B show the tumor-suppressing effect of the CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2-YTI on MC38-hCLDN18.2 xenografts, with solvent and TJ-CD4B used as controls. Figure 9A shows the tumor volume, and Figure 9B shows the mouse body weight.

Figure 10 shows the effect of CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2-YTI on mouse ALT/AST, with solvent and TJ-CD4B as controls.

Figure 11 shows the pharmacokinetic curve of CLDN18.2/4-1BB multifunctional antibody 1903x C5_V2 in h4-1BB transgenic mice, with 1903 as a control.

Detailed Implementation

The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

The preparation and purification methods of CLDN18.2/4-1BB binding protein in this application have been described in international patent application WO2024114676, and the entire contents of the aforementioned application documents can be incorporated into this disclosure.

The proteins used in this disclosure are: human 4-1BB protein (Human 4-1BB/TNFSF9 Protein, His Tag, purchased from Acrobiosystems, catalog number 41B-H5227), human 4-1BB protein (Biotinylated Human 4-1BB/TNFRSF9 Protein, Avitag ^™ , His Tag, purchased from Acrobiosystems, catalog number 41B-H82E3), and monkey 4-1BB protein (Cynomolgus/Rhesus macaque4-1BB/TNFRSF9 Protein, His Tag, purchased from Acrobiosystems, catalog number 41B-C52H4), with amino acid sequences ranging from Leu24 to Gln186.

The above protein reagents can be used in experiments of various embodiments of this disclosure, including as immunogenic antigens, screening antigens, and for activity and function identification. The amino acid sequences of the control molecules used in this disclosure are shown in Table 2.

Table 2. Control molecular sequences

Example 1. Screening and preparation of anti-4-1BB single-domain antibodies

1. Alpaca immunization, titer testing, and phage library affinity selection

Alpacas were immunized with His-tagged human 4-1BB recombinant protein (Acrobiosystems, 41B-H5227) four times, every two weeks. For the first immunization, 0.5 mg of antigen was mixed with 1 mL of Freund's complete adjuvant (CFA) and injected subcutaneously. For the subsequent three immunizations, 0.25 mg of antigen was mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. Blank serum was collected before immunization. One week after the third immunization and one week after the fourth immunization, 50 mL of peripheral blood was collected. PBMCs were isolated, total RNA was extracted, purity was determined, and the RNA was reverse transcribed into DNA. After two rounds of nested PCR, the nanobody target fragment was ligated to a phage display vector. Electroporation was performed to obtain a phage library.

Human 4-1BB protein sequence

To obtain anti-4-1BB nanobodies that simultaneously recognize both humans and monkeys, a two-round cross-screening strategy using human and monkey antigens was employed. The first and second rounds of screening used human 4-1BB and monkey 4-1BB, or monkey 4-1BB and human 4-1BB, respectively. Each round of screening employed Gly-HCl acid elution to elute phages specifically binding to 4-1BB. Ninety-six clones (a total of 192 clones) were randomly selected from the titer assay plates of both rounds. Positive clones were screened using phage ELISA, with the optical density measured at 450 nm. The positive clones were then sequenced. Based on the sequencing results, sequence alignment and phylogenetic analysis were performed, identifying 14 unique sequences, including H27, H170, C3, C5, and C145, with the C5 sequence shown below.

>C5

(Note: The underlined area is the CDR area)

Table 3. CDR (Kabat numbering rule) for anti-4-1BB single-domain antibody C5

2. Expression and purification of VHH-Fc fusion protein

The C5 sequence was ligated to human IgG1-Fc (SEQ ID NO: 16, underlined mutations) containing C220A, S267E, and L328F mutations (according to Eu system numbers). The resulting VHH-Fc fusion protein sequence is shown below. Furthermore, mutations such as L234A, L235A, and N297A (according to Eu system numbers) were introduced into the human IgG1 Fc to completely remove antibody FcγR-mediated effector function (e.g., as shown in SEQ ID NO: 15); and S228P (according to Eu system numbers) was introduced into the human IgG4 Fc to stabilize the antibody molecule and prevent half-molecule formation (e.g., as shown in SEQ ID NO: 16). These are all optional IgG Fc types. In SEQ ID NO: 14-16, underlined Fc mutations are indicated.

Human IgG1-Fc (containing C220A, S267E, and L328F mutations)

Human IgG1-Fc (containing C220A, L234A, L235A, and N297A mutations)

Human IgG4-Fc (containing the S228P mutation)

An example is listed as the antibody sequence C5 linked to SEQ ID NO: 14, as shown in SEQ ID NO: 17.

>C5-Fc


(Note: Italics are Fc)

A plasmid was constructed, transiently transfected into cells, and the antibody was expressed and purified. The target antibody was then obtained upon testing.

Example 2. Detection of antigen-binding activity and agonist activity of anti-4-1BB antibody

1. Detection of binding ability with 4-1BB antigen

The binding activity of the anti-4-1BB single-domain antibody to human 4-1BB protein was detected by flow cytometry.

HEK293-Hu4-1BB cells were obtained by transient transfection of HEK293 cells (ATCC CRL-1573) expressing human 4-1BB protein (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat#HG10041-ACR). The cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate-buffered saline, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB cells were washed twice with experimental culture medium. ^One × 10⁵ HEK293-Hu4-1BB cells were seeded per well in a 96-well U-plate. Different concentrations of the anti-4-1BB antibody VHH-Fc sample were added. After incubating the cells at 4°C for 1 hour, they were washed twice with experimental culture medium. Subsequently, goat anti-human IgG (H+L) Alexa Fluor 488 antibody (Thermo, Cat#A11013) was added, and after two washes, the fluorescence signal values were read by flow cytometry. Urelumab monoclonal antibody was used as a positive control. The MFI values of each antibody are shown in Figure 1.

The results showed that the anti-4-1BB antibodies involved had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, among which C5 had good cell membrane surface antigen binding activity.

2. Detection of agonist activity in the 4-1BB signaling pathway

The agonist activity of anti-4-1BB antibodies was assessed using the 4-1BB/NF-κB reporter gene.

HEK293 cells (ATCC CRL-1573) were transiently transfected with human 4-1BB gene (CD137cDNA ORF Clone, Human, C-OFPSpark tag; purchased from Sino Biological, Cat#HG10041-ACR) and NF-κB reporter gene (pGL4.32[luc2P/NF-κB-RE/Hygro] Vector, purchased from Promega, Cat#E849A) to obtain HEK293-Hu4-1BB/NF-κB double-transfected cells. The activation level of the NF-κB signaling pathway can be used to characterize 4-1BB activation. HEK293 cells were transiently transfected with FcγRIIb plasmid (CD32B/Fcgr2b cDNA ORF Clone, Human, N-His tag; purchased from Sino Biological, Cat#HG10259-NH) to obtain HEK293 cells with high expression of FcγRIIb. The cell culture medium was DMEM (Gibco, Cat#11995065) containing 10% fetal bovine serum. HEK293-Hu4-1BB/NF-κB cells (2× ^10⁶ /mL) were seeded at 50 μL into 96-well cell culture plates, followed by 40 μL of culture medium or HEK293 cells expressing FcγRIIb (2.5× ^10⁶ /mL). 10×10 μL of serially diluted anti-4-1BB antibody was added to each well, and the cells were incubated at 37°C for 6 hours. Cells were then removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) was added to each well. The cells were incubated in the dark for 5 minutes, and the fluorescence signal was measured using an Envision microplate reader (PerkinElmer, 2150). The _EC50 value and Emax value (fluorescence intensity relative to the antibody-free group) were calculated. The _EC50 value was used to evaluate the in vitro cell agonist activity of the anti-4-1BB antibody. The results are shown in Figure 2 and Table 4.

The results showed that without FcγRIIb (i.e., CD32b) positive cross-linking, the anti-4-1BB antibodies involved exhibited significantly weaker activation of the 4-1BB/NF-κB luciferase reporter gene signaling pathway compared to the Urelumab control, indicating that the disclosed anti-4-1BB antibody has better safety. After FcγRIIb cross-linking, the anti-4-1BB antibodies involved showed significant activation of the 4-1BB/NF-κB luciferase reporter gene signaling pathway, with C5 exhibiting the strongest activation ability comparable to the Urelumab control. Based on the combined activity results, sequence C5, which exhibits low background activation before FcγRIIb cross-linking and stronger activation activity after FcγRIIb cross-linking, was selected and humanized.

Table 4. _EC50 values of the agonistic activity of anti-4-1BB antibody against the 4-1BB/NF-κB luciferase reporter gene.

(Note: "-" indicates that the detected value exceeds the detection limit).

Example 3. Modification of anti-4-1BB antibody

1. Humanization transformation

Based on the Kabat numbering system, the CDR of the C5 sequence and the amino acid number of the FR region (human framework region) were used to identify the FR1, FR2, and FR3 sequences, which were then matched with antibody lineage databases to obtain highly homologous human FR lineage templates. FR1 was derived from IGHV3-64*04, FR2 from IGHV3-23*03, and FR3 from IGHV3-74*01. The FR regions of each human lineage were then inserted into the original sequences to reduce immunogenicity in humans. Reversal mutations were performed on key amino acids affecting antibody structure and function to restore binding affinity and activity. The humanized sequences are shown below.

>C5_V1

>C5_V2

>C5_V3

(Note: The underlined area is the CDR area)

The three humanized sequences were ligated into human IgG1-Fc (SEQ ID NO: 14). Plasmids were constructed, transiently transfected, expressed, and purified. The specific procedure was as follows: 60 μL of transfection reagent was diluted with culture medium and mixed with 15 μg of plasmid. The mixture was incubated at 37°C for 15 minutes. The transfection solution was then added dropwise to 30 mL of cell culture medium, and the cells were cultured on a shaker for one week. The supernatant was collected. Protein A affinity purification was then performed, eluted with citrate buffer (pH 3.4), and finally dialyzed with 1xPBS buffer before freezing.

2. Removal of TCE sites

Subsequently, we performed T-cell epitope (TCE) prediction on the C5_V2 sequence. Based on the prediction results, we modified the CDR3 sequence of C5_V2 to reduce the number of TCEs. The F mutation in the CDR3 region was changed to Y (F99Y, according to the Kabat numbering system) to obtain the TCE-optimized C5_V2 sequence, named C5_V2-YTI, whose sequence is as follows:

>C5_V2-YTI

That is, according to the Kabat numbering system, the amino acid sequence of CDR1 in C5_V2-YTI is shown as SEQ ID NO: 11, the amino acid sequence of CDR2 is shown as SEQ ID NO: 12, and the amino acid sequence of CDR3 is shown as HPLTYTIATMNDYDY (SEQ ID NO: 22).

The C5_V2-YTI sequence was ligated to human IgG1-Fc (SEQ ID NO: 14). The plasmid was constructed, transiently transfected, expressed, and purified.

3. Amino acid modification of the VH framework (FR) of the anti-4-1BB single-domain antibody: The entire text is introduced into WO2023093899 to obtain C5_V2-YTI-AA (corresponding to C5_V2-YTI-53 shown in sequence 30 of WO2023093899).

Example 4. Detection of antigen-binding activity and agonist activity of the modified anti-4-1BB antibody

1. Detection of binding ability with 4-1BB antigen

The antigen-binding activity of the humanized antibody was detected using the FACS assay described in Example 2. The results are shown in Table 5.

The results showed that the humanized anti-4-1BB antibodies involved had different degrees of binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, and C5_V1, C5_V2 and C5_V3 all maintained good binding activity.

Table 5. Affinity ( _EC50 values) of anti-4-1BB antibodies to human 4-1BB-overexpressing cell lines before/after humanization

2. Detection of agonist activity in the 4-1BB signaling pathway

The humanized antibody was used to detect 4-1BB/NF-κB signal activation dependent on the NF-κB luciferase reporter gene assay as described in Example 2. The results are shown in Figure 3 and Table 6.

The results showed that after cross-linking with FcγRIIb, the humanized anti-4-1BB antibody involved activated the 4-1BB/NF-κB luciferase reporter gene signaling pathway, and the activation capacity of C5, C5_V1, C5_V2, and C5_V3 was comparable to that of the Urelumab control.

Table 6. Activation of the NF-κB signaling pathway by anti-4-1BB antibodies before and after humanization


(Note: For Emax changes of less than 1.5, activity is defined as "-"; 1.5 to 2, it is defined as "+"; 2 to 2.5, it is defined as "++"; 2.5)
A value up to 3 is defined as "++"; a value greater than 3 is defined as "++++".

3. Detection of the binding ability of humanized 4-1BB antibody to antigen after TCE removal.

The humanized antibody C5_V2 was optimized by removing TCE, resulting in the antibody C5_V2-YTI (see Example 3). The antigen-binding activities of C5_V2 and C5_V2-YTI were compared using the FACS detection method described in Example 2, and the results are shown in Table 7. The results show that the C5_V2-YTI maintained good binding activity to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to that of C5_V2.

Table 7. Affinity _EC50 values of C5_V2, C5_V2-YTI with human 4-1BB highly expressed cell lines

Example 5. Preparation of CLDN18.2/4-1BB multifunctional antibody

1. Design, expression, and purification of CLDN18.2/4-1BB bispecific antibody (CLDN18.2x 4-1BB)

Based on the screening results of the humanized anti-4-1BB nanobodies, the C5_V2 clone was selected and used with the CLDN18.2 monoclonal antibody sequence (derived from WO2020200196A1) to construct a CLDN18.2 x 4-1BB bispecific antibody. The sequence of CLDN18.2 monoclonal antibody 1903 is as follows:

>CLDN18.2 monoclonal antibody 1903 heavy chain (SEQ ID NO: 23)

>CLDN18.2 monoclonal antibody 1903 light chain (SEQ ID NO: 24)

In the heavy chain, the italicized section represents the constant region of the heavy chain, while in the light chain, the italicized section represents the constant region of the light chain.

According to the Kabat numbering rules, the CDR for 1903 is:

HCDR1 is SYWMH (SEQ ID NO: 30), HCDR2 is MIHPNSGSTNYNEKFKG (SEQ ID NO: 31), and HCDR3 is LKTGNSFDY (SEQ ID NO: 32);

LCDR1 is KSSQSLLNSGNQKNYLT (SEQ ID NO: 33), LCDR2 is WASTRES (SEQ ID NO: 34), and LCDR3 is QNAYTYPFT (SEQ ID NO: 35).

The heavy and light chain variable regions and Fc segment sequences of the antibody are as follows:

VH of >1903 (SEQ ID NO: 36)

VL (SEQ ID NO: 37) of >1903

>IgG1 Fc (containing S239D, I332E mutation) (SEQ ID NO: 38)

The bispecific antibody molecule uses the aforementioned CLDN18.2 monoclonal antibody sequence as its backbone, with its Fc region carrying the S239D/I332E mutation. A C5_V2 molecule is fused to the C-terminus of each of the two heavy chains of CLDN18.2, using a (G ₄ S) ₂ linker, resulting in the bispecific antibody molecule 1903x C5_V2. Taking 1903x C5_V2 as an example, the naming convention for molecules in this disclosure is as follows: 1903 represents the variable region of the CLDN18.2 monoclonal antibody, and C5_V2 is the clone number of the 4-1BB nanobody. Replacing C5_V2 with C5_V2-YTI after removing the TCE yields the bispecific antibody molecule 1903x C5_V2-YTI, the antibody structure of which is shown in Figure 4. The above 1903x C5_V2-YTI bispecific antibody molecule was further optimized by modifying two amino acid residues at the C-terminus of C5_V2-YTI to remove pre-ADA. The optimized bispecific antibody molecule was named 1903x C5_V2-YTI-AA.

The amino acid sequence of the aforementioned bispecific antibody molecule is shown below:

>1903x C5_V2 heavy chain (SEQ ID NO: 25)

>1903x C5_V2-YTI heavy chain (SEQ ID NO: 26)

>1903x C5_V2-YTI-AA heavy chain (SEQ ID NO: 27)

In the above heavy chains, italics represent constant regions of the heavy chain, and underlined parts represent linkers. The light chain amino acid sequences of 1903x C5_V2, 1903xC5_V2-YTI, and 1903x C5_V2-YTI-AA are all shown in SEQ ID NO: 24.

Transient transfection and expression: Taking a 30mL expression system as an example, dilute the culture medium with 60μL of transfection reagent and mix with 15μg of plasmid. Incubate at 37℃ for 15 minutes, then add the mixed transfection solution dropwise while shaking the cell solution. Incubate on a shaker for one week, collect the supernatant, and centrifuge at 8000rpm for 5 minutes.

Antibody purification: First, equilibrate the Protein A affinity chromatography column with 1xPBS at a flow rate of 1 mL/min for 20 mL; load the sample at a flow rate of 1 mL/min; wash with 1xPBS at a flow rate of 1 mL/min for 20 mL; elute with citrate buffer (pH 3.4) at a flow rate of 1 mL/min, collect the elution in separate tubes, and read the absorbance at 280 nm using a NanoDrop spectrometer; finally, transfer the high-concentration protein to a dialysis bag and dialyze it into a beaker containing 1xPBS. The target antibody molecule was obtained upon detection.

Example 6. Antigen binding activity of C5_V2-YTI

We compared the antigen-binding activities of C5_V2, 1903x C5_V2-LALA, and 1903x C5_V2-YTI-LALA against human 4-1BB. C5_V2 was linked to human IgG1-Fc carrying the C220A/S267E/L328F mutation, as shown in SEQ ID NO: 17. 1903x C5_V2-LALA was obtained by introducing the L234A/L235A mutation into the Fc of the 1903xC5_V2 heavy chain (SEQ ID NO: 25) to obtain the heavy chain, and the Fc did not carry the S239D/I332E mutation (SEQ ID NO: 28), while the light chain remained as shown in SEQ ID NO: 24. 1903xC5_V2-YTI-LALA is obtained by introducing the L234A/L235A mutation into the Fc region of the 1903x C5_V2-YTI heavy chain (SEQ ID NO: 26), and the Fc region does not carry the S239D/I332E mutation (SEQ ID NO: 29). The light chain remains as shown in SEQ ID NO: 24. The sequence is as follows:

>1903x C5_V2-LALA heavy chain (SEQ ID NO: 28)

>1903x C5_V2-YTI-LALA heavy chain (SEQ ID NO: 29)

In the above heavy chains, italics represent constant regions of the heavy chain, and underlined parts represent connectors. The light chain sequences of 1903x C5_V2-LALA and 1903x C5_V2-YTI-LALA are both shown in SEQ ID NO: 24.

The binding activity of the C5_V2-YTI modified antibody to human 4-1BB protein was detected using FACS assays. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg/mL hygromycin B. After resuscitation, the cells were passaged to adjust their state. The experimental medium was sterile PBS (phosphate-buffered saline, pH 7.40) containing 2% fetal bovine serum. HEK293-Hu4-1BB cells were washed twice with experimental culture medium. 1 × ^10⁵ cells per well were seeded in a 96-well plate. Different concentrations of the test sample were added, and the cells were incubated at 4°C for 1 hour, followed by two washes with experimental culture medium. Then, Alexa Fluor 647-mouse anti-human (IgG, Fcγfragment specific) antibody (Jackson, Cat#209-605-098) was added, and after two washes, the fluorescence signal values were read by flow cytometry. The results are shown in Table 8.

FACS results showed that the 1903x C5_V2-YTI-LALA antibody had a strong binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to that of 1903x C5_V2-LALA and C5V2, indicating that C5_V2 and C5_V2-YTI have the same binding ability to 4-1BB.

Table 8. Results of antibody binding to HEK293-Hu4-1BB cells after C5_V2-YTI modification.

(Note: "-" indicates that the detection limit has been exceeded)

Example 7. Detection experiment of 4-1BB/NF-κB luciferase reporter gene of C5_V2-YTI

The agonist activity of the antibody was assessed using a 4-1BB/NF-κB reporter gene. HEK293 cells (ATCC CRL-1573) were transiently transfected with human 4-1BB gene (CD137 cDNA ORF Clone, Human, C-OFPSpark tag; Sino Biological, Cat#HG10041-ACR) and NF-κB genome (pGL4.32[luc2P/NF-κB-RE/Hygro]Vector, Promega, Cat#E849A) to obtain HEK293-Hu4-1BB/NF-κB double-transfected cells. The activation level of Hu4-1BB could be characterized by the activation level of the NF-κB signaling pathway. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells with human CLDN18.2 protein. The cell culture medium was RPMI 1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg/mL puromycin. HEK293-Hu4-1BB/NF-κB cells (2 × ^10⁶ /mL) were seeded at 50 μL into 96-well cell culture plates, and 40 μL of culture medium or NUGC4 cells expressing CLDN18.2 (2.5 × ^10⁶ /mL) were added. 10 × 10 μL of serially diluted test antibody was added to each well, and the plates were incubated at 37°C for 6 hours. Cells were removed, and an equal volume of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) was added to each well. Cells were incubated in the dark for 5 minutes, and the fluorescence signal was measured using an Envision microplate reader. The _EC50 and Emax values (fluorescence intensity relative to the antibody-free group) were calculated, and the in vitro cell agonist activity of the anti-4-1BB antibody was evaluated. Results are shown in Figure 5 and Table 9.

The results showed that the 1903x C5_V2-YTI-LALA antibody did not have background activation, but after cross-linking with CLDN18.2, its 4-1BB activating activity was comparable to that of 1903x C5_V2-LALA, indicating that C5_V2 and C5_V2-YTI have the same ability to activate 4-1BB.

Table 9. CLDN18.2-dependent 4-1BB/NF-κB luciferase reporter gene assay results of the C5_V2-YTI modified antibody.

Example 8. Detection of antigen-binding affinity of CLDN18.2/4-1BB multifunctional antibody

The affinity of the CLDN18.2/4-1BB multifunctional antibody for the antigen was detected using surface plasmon resonance (SPR) technology. The CM5 sensor chip was used, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P2O). Anti-human IgG (Fc) antibody was prepared into a 30 μg/mL solution using 10 mM sodium acetate buffer (pH 5.0), and the amino-coupling of the anti-human IgG (Fc) antibody channel was automatically performed using the Immobilization program. Each test antibody was prepared separately using HBS-EP+ buffer as a ligand to capture the anti-human IgG (Fc) antibody on the chip channel. Human 4-1BB protein (Acro Biosystems, 41B-H522a) and cynomolgus monkey 4-1BB protein (Acro Biosystems, 41B-C52H4) were used as antigens (i.e., analytes). The analytes were prepared using HBS-EP+ buffer, and serially diluted twofold. The diluted antibodies were flowed through the experimental and reference channels at a flow rate of 30 μL/min for 1 minute for binding and 15 minutes for dissociation. A 30-second run was performed using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) in regeneration buffer at a flow rate of 10 μL/min. Data were analyzed using a Zeba Spin Desalting Columns (Thermo, 89882) instrument and Biacore 8K evaluation software.

The results are shown in Table 10, which indicate that the antigen-binding affinity of the CLDN18.2x 4-1BB multifunctional antibody for human and cynomolgus monkey 4-1BB proteins is comparable to that of the parental 4-1BB VHH antibody C5_V2.

Table 10. Biacore affinity (1:1 binding) of CLDN18.2/4-1BB multifunctional antibody to 4-1BB

Example 9. Detection of antigen-binding activity of CLDN18.2/4-1BB multifunctional antibody

The binding activity of the CLDN18.2/4-1BB multifunctional antibody to human CLDN18.2/CD16A/4-1BB protein was detected using FACS assays. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells expressing human CLDN18.2 protein. The cell culture medium was RPMI 1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg/mL puromycin. CHO-K1-CD16A V176 cells were obtained by overexpressing human CD16A V176 protein in CHO-K1 cells. The cell culture medium was Ham's F12+GlutaMAX ^™ -I (Gibco, Cat#31765035) containing 10% fetal bovine serum and 200 μg/mL hygromycin B. HEK293-Hu4-1BB cells were obtained by overexpressing human 4-1BB protein in HEK293 cells. The cell culture medium was DMEM (Gibco, Cat#11965092) containing 10% fetal bovine serum and 100 μg/mL hygromycin B. After resuscitation, the cells were passaged to adjust their state. The experimental medium was sterile PBS (phosphate-buffered saline, pH 7.40) containing 2% fetal bovine serum. NUGC4-hi18.2, CHO-K1-CD16A V158, or HEK293-Hu4-1BB cells were washed twice with the experimental medium. 1 × ^10⁵ cells were seeded per well in a 96-well plate, and different concentrations of the test sample were added. After incubating the cells at 4°C for 1 hour, they were washed twice with the experimental medium. Subsequently, Alexa Fluor 647-mouse anti-human (IgG, Fcγfragment specific) antibody (Jackson, Cat#209-605-098) was added, and after two washes, the fluorescence signal values were read by flow cytometry. The results are shown in Tables 11 and 12.

FACS results showed that the CLDN18.2/4-1BB multifunctional antibody exhibited strong binding ability to CLDN18.2 on the surface of NUGC44-hi18.2 cells, comparable to that of CLDN18.2 monoclonal antibody 1903 and control antibody TJ-CD4B, and stronger than control antibody IMAB362. The CLDN18.2/4-1BB multifunctional antibody also exhibited strong binding ability to 4-1BB on the surface of HEK293-Hu4-1BB cells, comparable to 4-1BB monoclonal antibody C5_V2, and slightly stronger than TJ-CD4B. Among these, 1903x C5_V2 x 34H3 is another CLDN18.2/4-1BB multifunctional antibody obtained through screening in this application.

Table 11. Results of CLDN18.2/4-1BB multifunctional antibody binding to NUGC4-hi18.2 cells

Table 12. Results of CLDN18.2/4-1BB multifunctional antibody binding to HEK293-Hu4-1BB cells


(Note: "-" indicates that the detection limit has been exceeded)

Example 10. Experiment on the in vitro induction of antibody-mediated cell killing (ADCC) by CLDN18.2/4-1BB multifunctional antibody.

The antibody-mediated ADCC activity of NK cells against CLDN18.2-expressing target cells was assessed using a lactate dehydrogenase (LDH) assay. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells with human CLDN18.2 protein. The cell culture medium was RPMI 1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg/mL puromycin. SNU601 cells were purchased from Nanjing Kebai Biotechnology (CBP60507) and cultured in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum. Cryopreserved PBMCs were isolated from fresh human blood, thawed, and cultured overnight at 37°C in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum. The following day, different target cells were digested and resuspended in phenol red-free RPMI 1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum, and the density was adjusted to 2 × ^10⁵ cells/mL. Subsequently, 50 μL/well was seeded into 96-well plates, and 10 × 10 μL of serially diluted test antibody was added. The plates were incubated at 37°C in a 5% _CO₂ incubator for 0.5 hours.

PBMCs were collected and resuspended in phenol red-free RPMI 1640 containing 2% fetal bovine serum. The appropriate effector-to-target ratio was used according to the target cell type, and the cell density was adjusted accordingly. 40 μL/well was seeded into the above experimental plate and incubated at 37°C in a 5% _CO2 incubator for 4 hours. The cell culture plate was removed, and the cell culture supernatant was collected by centrifugation (400g, 5 minutes) using CytoTox. The Non-Radioactive Cytotoxicity Assay kit (Promega, G1780) is used to detect LDH levels. Refer to the kit's instruction manual for specific instructions.

The results are shown in Table 13, indicating that the CLDN18.2/4-1BB multifunctional antibody exhibited good ADCC activity against target cells with different CLDN18.2 expression levels, which was stronger than the control antibody IMAB362. Among them, SC190061(176F) is the PBMC donor number for the CD16A-F176 variant, and S2001102(176V) is the PBMC donor number for the CD16A-V176 variant.

Table 13. Results of NK cell killing of CLDN18.2-expressing target cells induced by CLDN18.2/4-1BB multifunctional antibody.

(Note: "-" indicates that the detection limit has been exceeded)

Example 11. Experiment on the in vitro induction of antibody-mediated phagocytosis (ADCP) by CLDN18.2/4-1BB multifunctional antibody.

PBMCs were isolated from fresh human blood, and then CD14+ monocytes were sorted using human CD14 microbeads (Miltenyi Biotec, 130-050-201). These CD14+ monocytes were cultured in macrophage differentiation medium RPMI1640 (Gibco, Cat#10491A-01) containing 50 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF, PeproTech, Cat#300-25) and 10% fetal bovine serum. After 6 days of differentiation, the macrophages became adhesive and developed tentacles. The macrophages were digested with trypsin for 5 minutes, gently scraped off with a spatula, and resuspended in RPMI1640 medium containing 10% fetal bovine serum, with the density adjusted to 4 × ^10⁵ cells/mL. They were then seeded into 96-well plates and incubated overnight at 37°C, 100 μL/well. The following day, NUGC4-hi18.2 cells were labeled with CellTrace Far Red (Invitrogen, C34564) at 37°C for 15 minutes. After washing twice with PBS, NUGC4-hi18.2 cells were added to wells already seeded with macrophages at a ratio of 50 μL/well (5 NUGC4-hi18.2 cells per macrophage). 4 × 50 μL of serially diluted test antibody was added to each well, and the target cells were subjected to phagocytosis for 4 hours. After phagocytosis, the cells were washed three times with PBS, and then stained with FITC anti-human/mouse CD11b (Tonbo, 35-0112-M100) for 30 minutes. After washing twice with PBS, the cells were analyzed by flow cytometry. Phagocytosis was measured by assessing the percentage of Far Red+/CD11b+ double-positive cells after gating CD11b-positive cells.

The results are shown in Table 14, which indicate that the CLDN18.2/4-1BB multifunctional antibody involved has good ADCP function, which is stronger than that of the control antibody IMAB362.

Table 14. Results of macrophage phagocytosis of tumor cells induced by CLDN18.2/4-1BB multifunctional antibody.

(Note: "-" indicates that the detection limit has been exceeded)

Example 12. CLDN18.2-dependent 4-1BB/NF-κB luciferase reporter gene assay of CLDN18.2/4-1BB multifunctional antibody.

4-1BB/NF-κB signaling activation dependent on CLDN18.2 was detected in the NF-κB luciferase reporter gene assay as described in Example 8.

The results are shown in Figure 6 and Table 15, indicating that the CLDN18.2/4-1BB multifunctional antibody involved did not exhibit background activation, and the agonistic activity of the CLDN18.2-dependent 4-1BB/NF-κB signaling pathway was comparable to that of TJ-CD4B. Among them, 1903x C5_V2 x 34H3 is another CLDN18.2/4-1BB multifunctional antibody obtained through screening in this application.

Table 15. Results of CLDN18.2-dependent 4-1BB/NF-κB luciferase reporter gene assay for CLDN18.2/4-1BB multifunctional antibody.


(Note: "-" indicates that the detection limit has been exceeded)

Example 13. T lymphocyte activation experiment

Freshly isolated and purified PBMCs were resuspended in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum, and the density was adjusted to 1× ^10⁶ cells/mL. NUGC4-hi18.2 cells were obtained by stably transfecting NUGC4 cells with human CLDN18.2 protein expression. The cell culture medium was RPMI 1640 (Gibco, Cat#10491A-01) containing 10% inactivated fetal bovine serum and 10 μg/mL puromycin. After resuscitation, the cells were passaged and the density was adjusted to 1.25× ^10⁶ cells/mL. 96-well plates were coated with 0.25 μg/mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85), 100 μL per well, and incubated at 37°C for 2 hours. Residual antibody was washed away with PBS. Subsequently, 100 μL of PBMC cells (1× ^10⁵ cells/well) and 80 μL of culture medium or NUGC4-hi18.2 cells (1× ^10⁵ cells/well) were seeded into OKT3-coated 96-well plates. 20 μL of different concentrations of the test sample were added to each well, and the plates were incubated at 37°C and 5% _CO₂ for 3 days. The cell culture plates were then removed, centrifuged (400g, 5 minutes), and the cell culture supernatant was collected. IL-2 levels were detected using a human IL-2 assay kit (Cisbio, Cat#62HIL02PEG). Refer to the kit's instructions for specific procedures.

The results are shown in Figures 7A and 7B, indicating that in both PBMC donors, after CLDN18.2 crosslinking, the CLDN18.2/4-1BB multifunctional antibodies involved could activate IL-2 secretion, comparable to the control antibody TJ-CD4B. Among them, 1903x C5_V2 x 34H3 is another CLDN18.2/4-1BB multifunctional antibody obtained in this application.

Example 14. Experiment on NK cell killing of T cells

PBMCs were isolated from fresh human blood, and T cells were sorted using the EasySep ^™ Human T Cell Enrichment Kit (Stemcell, Cat#17951). T75 culture flasks (Corning, Cat#430641) were coated with 0.25 μg/mL anti-CD3 antibody OKT3 (Invitrogen, Cat#16-0037-85) and incubated at 37°C for 2 hours. Residual antibody was washed away with PBS. Freshly isolated T cells were resuspended in RPMI 1640 (Gibco, Cat#10491A-01) containing 10% fetal bovine serum and seeded into OKT3-coated T75 culture flasks at a density adjusted to 2 × ^10⁶ cells/mL. After 48 hours of incubation, activated T cells were labeled with CellTrace Far Red (Invitrogen, C34564) at 37°C for 15 minutes. After washing twice with PBS, the cells were resuspended in phenol red-free RPMI 1640 (Gibco, Cat#11835-030) containing 2% fetal bovine serum, and the density was adjusted to 2 × ^10⁵ cells/mL. Subsequently, 50 μL/well was seeded into 96-well plates, and 10 × 10 μL of serially diluted test antibody was added. The plates were incubated at 37°C in a 5% _CO₂ incubator for 0.5 hours.

Freshly resuscitated PBMCs from the same donor were collected and resuspended in phenol red-free RPMI 1640 containing 2% fetal bovine serum. Cell density was adjusted according to an effector-to-target ratio of 25:1 (PBMC:activated T cells). 40 μL of fresh PBMCs were seeded per well in the above experimental plates and incubated at 37°C in a 5% _CO2 incubator for 6 hours. After incubation, the cell culture plates were removed, and PI (Absin, abs9358) was added at a specific ratio for staining for 10 minutes. After washing twice with PBS, flow cytometry was used for analysis. The cytotoxic effect of NK cells on activated T cells in PBMCs was measured by assessing the percentage of PI+/Far Red+ double-positive cells after CellTrace Far Red positive gating.

The results are shown in Figure 8, indicating that the CLDN18.2x 4-1BB bispecific antibody 1903x C5_V2-YTI did not induce NK cell killing of T cells, which was comparable to the isotype control IgG1. Among them, 1903x C5_V2 x34H3 is another CLDN18.2/4-1BB multifunctional antibody obtained in this application.

Example 15. Pharmacodynamics, Pharmacokinetics and Safety Evaluation

1. In vivo efficacy study of CLDN18.2/4-1BB multifunctional antibody in mouse colon cancer model MC38-hCLDN18.2

MC38-hCLDN18.2 cells (provided by Biocytogen) were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at a rate of 2 × ^10⁵ cells/100 μL/mouse. When the tumors grew to approximately 10² ^mm³ , 24 mice were randomly selected based on tumor volume and divided into four groups of six: solvent, 1903x C5_V2-YTI (0.26 mg/kg), and TJ-CD4B (0.3 mg/kg). The drugs were administered every two days. Tumor volume was measured twice weekly during the treatment and observation periods, and the measurements were recorded.

The tumor volume (TV) is calculated using the formula TV = 1/2 × a × ^b² , where a and b represent the long and short diameters of the tumor, respectively.

Relative tumor proliferation rate T/C% = (TT ₀ )/(CC ₀ )×100%; Tumor inhibition rate TGI% = 1-T/C%.

CR% (Complete Tumor Regression Rate) = Number of mice with complete tumor regression (<102 mm3) / Number of mice enrolled.

The results are shown in Figure 9A and Table 16, indicating that the CLDN18.2/4-1BB multifunctional antibody involved has good antitumor activity, which is superior to the control antibody TJ-CD4B. Among them, 1903x C5_V2 x 34H3 is another CLDN18.2/4-1BB multifunctional antibody obtained in this application.

Table 16. Antitumor effects of different CLDN18.2/4-1BB multifunctional antibodies on mouse xenografts

2. Toxicity assay of CLDN18.2/4-1BB multifunctional antibody in mouse colon cancer model MC38-hCLDN18.2

CLDN18.2/4-1BB multifunctional antibody cells (provided by Biocytogen) were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at a rate of 2 × ^10⁵ cells/100 μL/mouse. When the tumors grew to approximately 10² ^mm³ , 24 mice were randomly selected based on tumor volume and divided into four groups of six: solvent, 1903x C5_V2-YTI (0.26 mg/kg), and TJ-CD4B (0.3 mg/kg). Administered the drugs every two days. Mouse body weight was measured twice weekly during the administration and observation period, and the measurements were recorded. Blood samples were collected on day 17 to measure AST and ALT.

The results are shown in Figures 9B and 10. The results showed that the mice maintained stable body weight during administration, suggesting that the involved CLDN18.2/4-1BB multifunctional antibody had no significant toxic side effects. ALT/AST tests on day 17 showed that the involved CLDN18.2/4-1BB multifunctional antibody did not exhibit significant hepatotoxicity at an effective antitumor dose. Among them, 1903x C5_V2 x 34H3 is another CLDN18.2/4-1BB multifunctional antibody screened in this application.

3. Single-dose pharmacokinetic study of CLDN18.2/4-1BB multifunctional antibody in mouse colon cancer model MC38-hCLDN18.2

MC38-hCLDN18.2 cells were subcutaneously inoculated into B-h4-1BB humanized mice (provided by Biocytogen) at a rate of 5 × ^10⁵ cells/100 μL/mouse. When the tumor grew to approximately 110 ^mm³ , six mice were randomly selected based on tumor volume and divided into two groups of three: 1903 (10 mg/kg) and 1903x C5_V2 (12 mg/kg). A single intravenous injection was administered.

Blood samples were collected from the mice at the following time points: 0.25h, 6h, 24h, 72h, 144h, 240h, 336h, 408h, and 504h after drug administration. Blood drug concentrations were detected using an ELISA method. Goat anti-human IgG, Fc (Rockland, 609-101-017), was coated onto the bottom of the plate and incubated at 4°C for 14-18 hours. After washing three times with 300μL PBS, blocking reagent containing 3% BSA was added, and the plate was incubated at room temperature for 1 hour. After washing three times with 300μL PBS, the serum to be tested was added, and the plate was incubated at room temperature for 2 hours. After washing three times with 300μL PBS, 50μL of goat anti-human IgG-HRP (BETHYL, A80-304P) solution was added to each well, the plate was sealed, and the plate was incubated at room temperature for 1 hour. The secondary antibody concentration was 1:20000, with a dilution of 1% BSA-PBS + 0.5% mouse serum.

The results are shown in Figure 11 and Table 17. The CLDN18.2/4-1BB multifunctional antibody exhibited pharmacokinetic parameters similar to those of the monoclonal antibody, primarily based on antibody distribution mediated by the CLDN18.2 end. Similar to the PK characteristics of the parental CLDN18.2 monoclonal antibody 1903 at equimolar doses, these results indicate that the mouse plasma concentrations of the CLDN18.2/4-1BB multifunctional antibody were not affected by the 4-1BB end, exhibiting typical pharmacokinetic characteristics of an antibody targeting CLDN18.2.

Table 17. Pharmacokinetic parameters of CLDN18.2/4-1BB multifunctional antibody in h4-1BB transgenic mice (mean ± standard deviation)

The CLDN18.2/anti-4-1BB multifunctional antibody in the following formulations is 1903x C5_V2-YTI-AA with heavy chain and light chain sequences of SEQ ID NO:27 and 24.

Example 16. Study on the buffer system of CLDN18.2/anti-4-1BB multifunctional antibody formulation

1. Acetate buffer system

Three different pH buffer systems (10 mM acetate-sodium acetate, pH 4.5, 5.0, and 5.5) were used to prepare three formulations of CLDN18.2/anti-4-1BB multifunctional antibody with a concentration of 10 mg/mL. The purity of SEC-HPCL, non-reduced CE-SDS, and iCIEF of the samples was determined. The stability of the formulations under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 1 month) conditions was investigated.

F1) 10mM acetate-sodium acetate, pH 4.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F2) 10mM acetate-sodium acetate, pH 5.0, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F3) 10mM acetate-sodium acetate, pH 5.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 18. Formulation SEC-HPLC, Non-reducing CE-SDS, and iCIEF (acetate)

2. Histidine buffer system

Three different pH buffer systems (10 mM histidine-histidine hydrochloride, pH 5.5, 6.0, and 6.5) were used to prepare three formulations of CLDN18.2/anti-4-1BB multifunctional antibody with a concentration of 10 mg/mL. The purity of SEC-HPCL, non-reduced CE-SDS, and iCIEF of the samples were determined. The stability of the three formulations under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 1 month) was investigated.

F4) 10mM histidine-histidine hydrochloride, pH 5.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F5) 10mM histidine-histidine hydrochloride, pH 6.0, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F6) 10mM histidine-histidine hydrochloride, pH 6.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 19. Formulation SEC-HPLC, Non-reducing CE-SDS, and iCIEF (histidine salt)

3. Citrate buffer system

Three different pH buffer systems (10 mM citrate-sodium citrate, pH 5.5, 6.0, and 6.5) were used to prepare three formulations of CLDN18.2/anti-4-1BB multifunctional antibody with a concentration of 10 mg/mL. The purity of SEC-HPCL, non-reduced CE-SDS, and iCIEF in the samples was determined. The stability of the three formulations under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 1 month) was investigated.

F7) 10mM citrate-sodium citrate, pH 5.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F8) 10mM citrate-sodium citrate, pH 6.0, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F9) 10mM citrate-sodium citrate, pH 6.5, 10mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 20. Formulation SEC-HPLC, Non-reducing CE-SDS, and iCIEF (citrate)

Note: ^a NT indicates not detected. Samples in the citrate system showed higher SEC-HPLC polymer concentrations at 0°C and after 7 days of light exposure; therefore,
Some samples under certain conditions (40℃ - January) were not tested.

The results show:

(1) After being placed under light for 7 days, the SEC-HPLC monomer percentage of the acetate antibody preparation and the histidine antibody preparation was relatively higher than that of the citrate buffer system.

(2) After being placed at a high temperature of 40℃ for 1 month, the NR-CE main peak purity and iCIEF main peak percentage of the antibody preparation containing histidine salt and pH 6.0-6.5 were relatively higher than those of the acetate buffer system.

Therefore, histidine buffer system is suitable as a buffer system for CLDN18.2/anti-4-1BB multifunctional antibody formulation.

Example 17. Screening of excipients for CLDN18.2/anti-4-1BB multifunctional antibody formulation

To further enhance the stability of the CLDN18.2/anti-4-1BB multifunctional antibody formulation under light and high temperature (e.g., 40°C) conditions, further screening of excipients is required.

A 10 mM histidine-histidine hydrochloride buffer system at pH 6.0 was used. The surfactant was 0.02% (w/v) polysorbate 80. CLDN18.2/anti-4-1BB multifunctional antibody at a concentration of 20 mg/mL was used, along with different excipients to prepare antibody formulations. The excipients were 8% (w/v) sucrose, 8% (w/v) sucrose + 0.002% (w/v) disodium edetate, 8% (w/v) sucrose + 0.1% (w/v) methionine, 4.5% (w/v) sucrose + 0.5% (w/v) sodium chloride, and 2.5% (w/v) proline. The appearance, SEC-HPLC, non-reducing CE-SDS, and IEC purity of the samples were determined. The stability of each formulation was investigated under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 4 weeks). The formulation formulations are as follows:

F10) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F11) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F12) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.1% (w/v) methionine, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F13) 10mM histidine-histidine hydrochloride, 4.5% (w/v) sucrose, 0.5% (w/v) sodium chloride, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F14) 10mM histidine-histidine hydrochloride, 2.5% (w/v) proline, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 21. Appearance of the formulation

Table 22. Formulation SEC-HPLC, Non-reducing CE-SDS and IEC

Note: The detection method for ^beta -charged heterostructures has been changed from iCIEF to IEC. NT indicates not detected.

The results show:

(1) From the appearance of the formulation, the antibody formulation containing sodium chloride excipient showed a micro-emulsification under conditions of 0°C, 7 days of light exposure, and 40°C for 4 weeks. In contrast, the formulation containing other excipients showed a clear appearance with no visible protein particles. Therefore, antibody formulations containing sucrose (and its combination with disodium edetate, methionine, or sodium chloride) or proline have slightly better appearance stability than antibody formulations containing sodium chloride excipient.

(2) Under 7 days of illumination, there was no significant difference in the percentage of the main peak area of each formulation in IEC. Compared with antibody formulations containing other excipients, antibody formulations containing disodium edetate excipients had relatively higher SEC-HPLC monomer percentage and non-reduced CE-SDS main peak under high temperature (40℃-4 weeks) conditions.

Therefore, sucrose plus disodium edetate is suitable as an excipient for the CLDN18.2/anti-4-1BB multifunctional antibody formulation.

Example 18. Study on the concentration of polysorbate 80 in CLDN18.2/anti-4-1BB multifunctional antibody formulation

A 10 mM histidine-histidine hydrochloride buffer system at pH 6.0 was used to prepare the antibody formulation with a concentration of 20 mg/mL of CLDN18.2/anti-4-1BB multifunctional antibody. The excipient composition was 8% (w/v) sucrose + 0.002% (w/v) disodium edetate and different concentrations of surfactants. The surfactants were 0.06% (w/v) polysorbate 80, 0.04% (w/v) polysorbate 80, and 0.02% (w/v) polysorbate 80. The purity of the samples was determined by SEC-HPLC and IEC. The stability of each formulation was investigated under freeze-thaw cycles (-35℃/RT, 5 times), shaking (200 rpm, 25℃, for 7 days), and high temperature (40℃, for 4 weeks).

F15) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.06% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F16) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.04% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F17) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.02% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 23. Formulation SEC-HPLC and IEC

Note: ^c N/A indicates not applicable. ^d NT indicates not detected.

The results show:

After freeze-thaw cycles, shaking, and/or high-temperature (40°C for 4 weeks) storage, formulations of polysorbate 80 at different concentrations showed no significant differences in SEC-HPLC and IEC purity under the same experimental conditions. Therefore, 0.02% w/v–0.06% w/v polysorbate 80 is suitable for CLDN18.2/anti-4-1BB multifunctional antibody formulations.

Example 19. CLDN18.2/Anti-4-1BB Multifunctional Antibody Concentration Screening

Different concentrations of CLDN18.2/anti-4-1BB multifunctional antibodies (20 mg/mL, 30 mg/mL) were prepared using a 10 mM histidine-histidine hydrochloride buffer system at pH 6.0. The excipient composition was 8% (w/v) sucrose + 0.002% (w/v) disodium edetate, and the surfactant was 0.04% (w/v) polysorbate 80. The appearance, purity by SEC-HPLC and non-reducing CE-SDS were determined. The stability of each formulation under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 2 and 4 weeks) was investigated.

F18) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.04% (w/v) polysorbate 80, pH 6.0, 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F19) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.04% (w/v) polysorbate 80, pH 6.0, 30 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 24. Formulation SEC-HPLC and Non-reducing CE-SDS

Example 20. Screening of excipients for liquid formulations

The CLDN18.2/anti-4-1BB multifunctional antibody with a concentration of 50 mg/mL was prepared using a 10 mM histidine-histidine hydrochloride buffer system at pH 6.0. The excipients consisted of 8% (w/v) sucrose and 8% (w/v) sucrose + 0.002% disodium edetate, with 0.06% (w/v) polysorbate 80 as the surfactant. The purity of the samples was determined by SEC-HPLC, non-reducing CE-SDS, and IEC. The stability of each formulation was investigated under freeze-thaw cycles (-35℃/RT, 5 cycles), -35℃ (for 1 month), shaking (200 rpm, 25℃, for 3 days), 2–8℃ (for 4 weeks), and room temperature light (450±50 lx, 25℃, for 3 days).

F20) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.06% (w/v) polysorbate 80, pH 6.0, 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F21) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% disodium edetate, 0.06% (w/v) polysorbate 80, pH 6.0, 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 25. Results of excipient screening for liquid dosage forms 1 2

Note: ^e N/A indicates not applicable.

The results show:

Under the same experimental conditions, there were no significant differences in stability among the formulations. When the antibody concentration was 50 mg/mL, there were no significant differences in the stability of the liquid formulations composed of "sucrose + polysorbate 80" and "sucrose + sodium edetate + polysorbate 80" during long-term storage, transportation, and formulation production.

Example 21. Screening of excipients for lyophilized formulations

A liquid formulation with an antibody concentration of 50 mg/mL was selected and prepared into a lyophilized formulation.

A 10 mM histidine-histidine hydrochloride buffer system at pH 6.0 was used to prepare a formulation of CLDN18.2/anti-4-1BB multifunctional antibody with a concentration of 50 mg/mL. The excipients consisted of either 8% (w/v) sucrose or 8% (w/v) sucrose + 0.002% disodium edetate, and the surfactant was 0.06% (w/v) polysorbate 80. The formulation was then lyophilized (including pre-freezing, primary drying, and secondary drying). The reconstitution time, appearance of the reconstituted solution, SEC-HPCL, non-reducing CE-SDS, and IEC purity of the lyophilized formulation were determined. The stability of the formulation under light (4500±500 lx, 25℃, for 7 days) and high temperature (40℃, for 2 and 4 weeks) conditions was investigated.

F22) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.06% (w/v) polysorbate 80, pH 6.0, 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

F23) 10mM histidine-histidine hydrochloride, 8% (w/v) sucrose, 0.002% disodium edetate, 0.06% (w/v) polysorbate 80, pH 6.0, 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody

Table 26. SEC-HPLC, Non-reducing CE-SDS and IEC of Lyophilized Formulations

The results show:

Compared with the initial value of 0, under different stability test conditions, the two groups of lyophilized formulations did not show significant changes in reconstitution time, appearance of the reconstituted formulations, or purity, indicating that the formulations of both groups have good stability.

Example 22. Liquid formulation of CLDN18.2/anti-4-1BB multifunctional antibody (optional)

This invention provides a liquid formulation comprising, but not limited to, “20-40 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.02%-0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride, pH 6.0”, including but not limited to:

1) 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.02% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

2) 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.04% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

3) 20 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

4) 30 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.02% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

5) 30 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.04% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

6) 30 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

Experimental results show that the above-mentioned CLDN18.2/anti-4-1BB multifunctional antibody preparations all have good stability and can be used in the formulation of CLDN18.2/anti-4-1BB multifunctional antibody drugs.

Example 23. Optional lyophilized formulation of CLDN18.2/anti-4-1BB multifunctional antibody

This invention provides a lyophilized formulation comprising, but not limited to, “20-50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, with or without 0.002% (w/v) disodium edetate, 0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride, pH 6.0”, including but not limited to:

7) 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0;

8) 50 mg/mL CLDN18.2/anti-4-1BB multifunctional antibody, 8% (w/v) sucrose, 0.002% (w/v) disodium edetate, 0.06% (w/v) polysorbate 80, 10 mM histidine-histidine hydrochloride pH 6.0.

Experimental results show that the above-mentioned CLDN18.2/anti-4-1BB multifunctional antibody preparations all have good stability and can be used in the formulation of CLDN18.2/anti-4-1BB multifunctional antibody drugs.

While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure.

Claims (21)

A pharmaceutical composition comprising: a) CLDN18.2/4-1BB binding protein, which includes: Specifically binds to the first antigen-binding domain of 4-1BB; and It specifically binds to the second antigen-binding domain of CLDN18.2. Wherein, the first antigen-binding domain includes an immunoglobulin single variable domain, and the immunoglobulin single variable domain includes: CDR1, CDR2, and CDR3 in any of the amino acid sequences shown in SEQ ID NO: 10, 18-21, wherein CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the single variable domain of the immunoglobulin are as shown in SEQ ID NO: 11, 12 and 13, or as shown in SEQ ID NO: 11, 12 and 22, respectively; and b) a buffer, wherein the buffer is selected from acetate buffer, citrate buffer and histidine buffer, preferably a histidine buffer, more preferably a histidine-hydrochloride buffer or a histidine-acetate buffer, and most preferably a histidine-histidine hydrochloride buffer. According to claim 1, the CLDN18.2/4-1BB binding protein is modified by humanizing the single variable domain of the immunoglobulin, maturing affinity, removing T cell epitopes, reducing antibody deamide and/or reducing antibody isomerization. Preferably, the heavy chain framework region of the humanoid template used in the humanization process is IGHV3-64*04, IGHV3-23*03 and/or IGHV3-74*01. According to claim 1 or 2, the CLDN18.2/4-1BB binding protein has an amino acid sequence of the single variable domain of the immunoglobulin as shown in any one of SEQ ID NO: 10, 18-21 or has at least 80% or at least 90% sequence identity with it. The CLDN18.2/4-1BB binding protein according to any one of claims 1 to 3, wherein, The second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH contains HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 36. The VL contains LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 37. The HCDR and LCDR are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 30, 31, and 32, and the VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 33, 34, and 35. The CLDN18.2/4-1BB binding protein according to claim 4, wherein the VH comprises an amino acid sequence as shown in or having at least 80% or at least 90% identity with SEQ ID NO: 36. The VL contains an amino acid sequence as shown in SEQ ID NO: 37 or having at least 80% or at least 90% identity with it. The CLDN18.2/4-1BB binding protein according to any one of claims 1 to 5 further comprises the Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, or human IgG4; More preferably, the Fc region is an effector-enhanced Fc region. According to claim 6, the CLDN18.2/4-1BB binding protein, when the Fc region is the Fc region of human IgG1, contains any one or any combination of the following amino acid mutations: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 301A; 332E; 239D/332E; 239D/330S/332E; 239D/330L/332E; 298A/333A/334A; 247I/339D; 247I/339 Q; 280H/290S; 280H/290S/298D; 280H/290S/298V; 243L/292P/300L; 243L/292P/300L/396L; 243L/292P/300L/305I/396L; 236A/239D/332E; 326A/333A; 326W/333S; 290E/298G/299A; 290N/298G/299A; 290E/298G/299A/326E; or 290N/298G/299A/326E; the mutations are defined according to the EU numbering system; Preferably, the Fc region contains any one or any combination of the following amino acid mutations: S239D; S239E; S239K; F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D/I332E; S239D/A330S/I332E; S239D/A330L/I332E; S298A/D333A/K334A; P247I/A339D; P247I/A339Q; D280H/K290S; D2 80H/K290S/S298D; D280H/K290S/S298V; F243L/R292P/Y300L; F243L/R292P/Y300L/P396L; F243L/R292P/Y300L/V305I/P396L; G236A/S239D/I332E; K326A/E333A; K326W/E333S; K290E/S298G/T299A; K290N/S298G/T299A; K290E/S298G/T299A/K326E; or K290N/S298G/T299A/K326E. The CLDN18.2/4-1BB binding protein according to any one of claims 1 to 7 further comprises a linker; Preferably, the amino acid sequence of the linker is as shown in ( _{GmSn ) h} or (GGNGT) _h or (YGNGT) _h or (EPKSS) _h , wherein m and n are each independently selected from integers 1-8, and h is independently selected from integers 1-20; More preferably, the connector is the connector shown in (G ₄ S) ₂ or (G ₄ S) ₃ . According to any one of claims 1 to 8, the CLDN18.2/4-1BB binding protein, the second antigen-binding domain comprises a heavy chain and a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 23 or has at least 80% to 90% sequence identity with it. The amino acid sequence of the light chain is as shown in SEQ ID NO: 24 or has at least 80% or at least 90% sequence identity with it. The CLDN18.2/4-1BB binding protein according to any one of claims 1 to 9, comprising a first polypeptide chain and a second polypeptide chain selected from the following: (1) The amino acid sequence of the first polypeptide chain is as shown in any of SEQ ID NO: 25-27 or has at least 80% or at least 90% sequence identity with it. The amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 24 or has at least 80% or at least 90% sequence identity with it. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pH of the composition is 4.5-7.5, preferably 4.8-7.2, more preferably 5.0-7.0, and most preferably 5.3-6.7. The pharmaceutical composition according to any one of claims 1 to 11, wherein the concentration of the buffer is 0.1-50 mM, preferably 0.5-40 mM, more preferably 1-30 mM, and most preferably 5-20 mM. The pharmaceutical composition according to any one of claims 1 to 12 further comprises one or more excipients selected from amino acids or their pharmaceutically acceptable salts, metal chelators, salts and sugars; Preferably, the amino acid is selected from one or more of methionine and proline; the sugar is selected from one or more of sucrose and trehalose; the metal chelating agent is edetic acid or its pharmaceutically acceptable salt; and the salt is sodium chloride. More preferably, the excipient is selected from one or more of sucrose, methionine or its pharmaceutically acceptable salt, proline or its pharmaceutically acceptable salt, edetic acid or its pharmaceutically acceptable salt, and sodium chloride; Most preferably, the excipient is sucrose, or sucrose and edemaic acid or its pharmaceutically acceptable salt. According to the pharmaceutical composition of claim 13, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.01% w/v to 20% w/v, preferably 0.05% w/v to 15% w/v, more preferably 0.08% w/v to 10% w/v, and most preferably 0.08% w/v to 5% w/v; The concentration of the metal chelating agent is 0.0001% w/v-0.5% w/v, preferably 0.0003% w/v-0.1% w/v, more preferably 0.0005% w/v-0.05% w/v, and most preferably 0.0008% w/v-0.01% w/v; The concentration of the salt is 0.005% w/v-15% w/v, preferably 0.01% w/v-10% w/v, more preferably 0.05% w/v-5% w/v, and most preferably 0.1% w/v-1% w/v; The sugar concentration is 0.05% w/v to 30% w/v, preferably 0.1% w/v to 20% w/v, more preferably 0.5% w/v to 15% w/v, and most preferably 1% w/v to 10% w/v. The pharmaceutical composition according to any one of claims 1 to 14 further comprises a surfactant, wherein the surfactant is preferably polysorbate, more preferably one or more of polysorbate 20 and polysorbate 80, and most preferably polysorbate 80. And/or, wherein the surfactant concentration is 0.001% w/v to 1% w/v, preferably 0.005% w/v to 1% w/v, more preferably 0.008% w/v to 0.2% w/v, and most preferably 0.01% w/v to 0.1% w/v. The pharmaceutical composition according to any one of claims 1 to 15, wherein the concentration of the CLDN18.2/4-1BB binding protein is 0.05-300 mg/mL, preferably 0.1-200 mg/mL, more preferably 0.5-100 mg/mL, and most preferably 1-80 mg/mL. A pharmaceutical composition comprising any one of the following: A) The CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; Histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably polysorbate 80; sucrose; B) CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; Histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably polysorbate 80; Sucrose and edetate or their pharmaceutically acceptable salts, preferably sucrose and disodium edetate; C) CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; Histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably polysorbate 80; Sucrose and methionine or their medicinal salts; D) CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; Histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably polysorbate 80; Sucrose and sodium chloride; E) CLDN18.2/4-1BB binding protein as claimed in any one of claims 1 to 10; Histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably polysorbate 80; Proline or its medicinal salts. Alternatively, the pharmaceutical composition comprises any one of the following groups: A-1) 0.05-300 mg/mL of CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 0.1-50mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.001% w/v - 1% w/v polysorbate, preferably polysorbate 80; 0.05% w/v - 30% w/v sucrose; The pH of the composition is 4.5-7.5; A-2) 0.1-200 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 0.5-40mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005% w/v - 1% w/v polysorbate, preferably polysorbate 80; 0.1% w/v - 20% w/v sucrose; The pH of the composition is 4.8-7.2; A-3) 0.5-100 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 1-30mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008% w/v - 0.2% w/v polysorbate, preferably polysorbate 80; 0.5% w/v - 15% w/v sucrose; The pH of the composition is 5.0-7.0; A-4) 1-80 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 5-20mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% w/v - 0.1% w/v polysorbate, preferably polysorbate 80; 1% w/v - 10% w/v sucrose; The pH of the composition is 5.3-6.7; B-1) 0.05-300 mg/mL of CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 0.1-50mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.001% w/v - 1% w/v polysorbate, preferably polysorbate 80; 0.05% w/v-30% w/v sucrose and 0.0001% w/v-0.5% w/v edetate or its pharmaceutically acceptable salt, preferably sucrose and disodium edetate; The pH of the composition is 4.5-7.5; B-2) 0.1-200 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 0.5-40mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005% w/v - 1% w/v polysorbate, preferably polysorbate 80; 0.1% w/v-20% w/v sucrose and 0.0003% w/v-0.1% w/v edetate or its pharmaceutically acceptable salt, preferably sucrose and disodium edetate; The pH of the composition is 4.8-7.2; B-3) 0.5-100 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 1-30mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008% w/v - 0.2% w/v polysorbate, preferably polysorbate 80; 0.5% w/v-15% w/v sucrose and 0.0005% w/v-0.05% w/v edetate or its pharmaceutically acceptable salt, preferably sucrose and disodium edetate; The pH of the composition is 5.0-7.0; B-4) 1-80 mg/mL CLDN18.2/4-1BB binding protein as described in any one of claims 1 to 10; 5-20mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% w/v - 0.1% w/v polysorbate, preferably polysorbate 80; 1% w/v-10% w/v sucrose and 0.0008% w/v-0.01% w/v edetate or its pharmaceutically acceptable salt, preferably sucrose and disodium edetate; The pH of the composition is 5.3-6.7. Alternatively, the pharmaceutical composition comprises any one of the following groups: A-5) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% w/v - 0.1% w/v polysorbate 80; Approximately 8% w/v sucrose; The pH of the composition is approximately 6.0; A-6) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.02% w/v polysorbate 80; Approximately 8% w/v sucrose; The pH of the composition is approximately 6.0; A-7) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.04% w/v polysorbate 80; Approximately 8% w/v sucrose; The pH of the composition is approximately 6.0; A-8) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.06% w/v polysorbate 80; Approximately 8% w/v sucrose; The pH of the composition is approximately 6.0; B-5) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% w/v - 0.1% w/v polysorbate 80; About 8% w/v sucrose and about 0.002% w/v edemaic acid or a pharmaceutically acceptable salt thereof, preferably disodium edemaic acid; The pH of the composition is approximately 6.0; B-6) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.02% w/v polysorbate 80; About 8% w/v sucrose and about 0.002% w/v edemaic acid or a pharmaceutically acceptable salt thereof, preferably disodium edemaic acid; The pH of the composition is approximately 6.0; B-7) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.04% w/v polysorbate 80; About 8% w/v sucrose and about 0.002% w/v edemaic acid or a pharmaceutically acceptable salt thereof, preferably disodium edemaic acid; The pH of the composition is approximately 6.0; B-8) about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL or about 80 mg/mL as claimed in any one of claims 1 to 10; Approximately 10 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Approximately 0.06% w/v polysorbate 80; About 8% w/v sucrose and about 0.002% w/v edemaic acid or a pharmaceutically acceptable salt thereof, preferably disodium edemaic acid; The pH of the composition is approximately 6.0. A lyophilized formulation, wherein the lyophilized formulation, upon reconstitution, can form the pharmaceutical composition according to any one of claims 1-17, or the lyophilized formulation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1-17. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized formulation of claim 18. An article comprising a container containing a pharmaceutical composition as described in any one of claims 1 to 17, a lyophilized formulation as described in claim 18, or a reconstituted solution as described in claim 19. Use of the pharmaceutical composition of any one of claims 1 to 17, the lyophilized formulation of claim 18, or the reconstituted solution of claim 19 in the preparation of a medicament for treating cancer; Preferably, 1) the cancer is CLDN18.2 positive; And/or 2) the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, or any combination thereof.