WO2023125729A1 - Anti-cd3 humanized antibody and application thereof in preparation of bispecific antibody - Google Patents

Abstract

Provided is an anti-CD3 antibody. The provided anti-CD3 antibody has a reduced affinity for the antigen CD3. Furthermore, after constructing the anti-CD3 antibody together with an antibody which targets other antigens so as to form a bispecific antibody, T cells can still be effectively recruited at tumor cell sites, thereby achieving an excellent treatment effect. Therefore, the provided anti-CD3 antibody or a fragment thereof can be used to construct a multispecific antibody, specifically a bispecific antibody, and has the effect of binding T cell surface CD3 so as to effectively recruit T cells without overstimulating T cells.

Description

A kind of anti-CD3 humanized antibody and its application in the preparation of bispecific antibody

Cross References to Related Applications

This patent application claims the priority right of the Chinese invention patent application with application number CN202111670032.9 filed on December 31, 2021, the entire contents of which are hereby incorporated by reference.

technical field

The invention belongs to the field of biomedicine, in particular, the invention relates to an anti-human CD3 humanized antibody or its functional fragment, and the application of the antibody or its functional fragment.

Background technique

CD3 molecule is mainly distributed on the surface of mature T lymphocytes, and is an important differentiation antigen on the membrane of T cells. Studies have shown that CD3 molecules are composed of at least five polypeptide chains: gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and Eta (η); among them, CD3-epsilon is related to CD3-gamma, CD3-delta and CD3-zeta and heterodimers of the T cell receptors alpha/beta and gamma/delta together constitute the T cell receptor (TCR)-CD3 complex. The TCR-CD3 complex plays an important role in coupling antigen recognition to intracellular signal transduction pathways: part of the TCR-CD3 complex exists on the surface of T lymphocytes, and when antigen-presenting cells (APCs) activate TCR, TCR-mediated The signal induced by the CD3 molecule is transmitted on the cell membrane through CD3-delta, CD3-epsilon, CD3-gamma and CD3-zeta, and all CD3 molecules contain an immunoreceptor tyrosine activation domain (ITAM) in their cytoplasmic domain, in Following TCR engagement, these domains are phosphorylated by the src family of protein tyrosine kinases LCK and FYN, leading to activation of downstream signaling pathways.

At present, many activating CD3 antibodies are sold clinically or in the pharmaceutical market, and the dosage of these activating CD3 antibodies is generally low, because if the dose of CD3 antibody drugs is too high, there will be excessive activation of T cells to produce cytokines The phenomenon of the storm presents a considerable safety risk. Therefore, the current research and development of antibody drugs has tended to seek antibodies with suitable affinity for antigens or targets to ensure the safety and efficacy of drugs.

Therefore, there is still a need in the art to provide an antibody with a suitable affinity for CD3.

Contents of the invention

In view of the above problems, the purpose of the present invention is to provide a novel antibody with appropriate affinity for CD3, especially CD3 on the surface of human T cells. Another object of the present invention is to provide the application of the anti-CD3 antibody or the functional regions contained therein (such as heavy chain variable region and light chain variable region) in the preparation of antibody medicine.

The present invention provides the following technical solutions.

In one aspect, the present invention provides an antibody or fragment thereof, which can specifically bind to CD3, especially CD3 on the surface of human T cells.

Specifically, the antibody or fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) ) comprising the following CDR combinations (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): HCDR-1 comprising the amino acid sequence (TYAMN) shown in SEQ ID NO: 22, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:23 (RIRSKYNNYATYYADSVKD), HCDR-3 comprising the amino acid sequence shown in SEQ ID NO:24 (HGNFGNSYVSYFAY); and, comprising the amino acid shown in SEQ ID NO:25 LCDR-1 of sequence (RSSTGAVTTSNYAN), LCDR-2 comprising the amino acid sequence (GTNKRAP) shown in SEQ ID NO:26, LCDR-3 comprising the amino acid sequence (ALWYSNLWV) shown in SEQ ID NO:27.

In the context of the present invention, the combination of light and heavy chain CDRs provided comes from the heavy chain and light chain amino acid sequences of the anti-CD3 antibody specifically described in the specific embodiments of the present invention. CDRs contained in the chain and light chain. For example, the combinations of light and heavy chain CDRs provided above were obtained using the KABAT definition method.

In the antibody or fragment thereof provided by the present invention, preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with the amino acid sequence; and/or Alternatively, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence at least 75% identical to said amino acid sequence.

In the context of the present invention, "at least 75% identity" is an identity of any percentage figure between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity.

In the context of the present invention, both the "heavy chain variable region" and the "light chain variable region" include the above-mentioned CDR combination and the spaced framework region (framework region), and the arrangement of each structural domain is: FR1-CDR1-FR2 - CDR2-FR3-CDR3-FR4. Further optionally, at most 25% difference in amino acid sequence resulting from said "at least 75% identity" may exist in any framework region in the heavy chain variable region or light chain variable region, or in the heavy chain variable region. In any domain or sequence other than the variable region and light chain variable region. The differences may result from amino acid deletions, additions or substitutions at any position.

As far as the antigen is concerned, the antibody or fragment thereof of the present invention is an anti-CD3 antibody or fragment thereof; preferably, the antibody is mouse anti, rabbit anti, monoclonal antibody, chimeric antibody, partially or fully humanized antibody etc., or the fragment is a half antibody or an antigen-binding fragment of an antibody or half antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; more preferably Preferably, the antibody is IgG. According to a specific embodiment of the present invention, the present invention provides an isolated and structurally characterized human antibody, such as a human monoclonal antibody, that specifically binds CD3, particularly CD3 on the surface of human T cells.

In addition to the variable region, the antibody or fragment thereof provided by the invention also comprises a constant region, such as a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or light chain constant region (CL ); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region and/or a kappa or lambda type of IgG, IgA, IgM, IgD or IgE Light chain constant region.

According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. Preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype, and the light chain constant region is of κ type. Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence at least 75% identical to the amino acid sequence shown in SEQ ID NO: 17; the monoclonal antibody The light chain constant region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 18.

According to a specific embodiment of the present invention, the anti-human CD3 antibody of the present invention is a monoclonal antibody. Preferably, the anti-human CD3 antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is of human IgG1 subtype.

On the other hand, the present invention also provides a nucleic acid molecule, which comprises a heavy chain CDR, light chain CDR, light chain variable region, heavy chain variable region, heavy chain or Nucleotide sequence of the light chain.

The nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Thus in a further aspect, the invention also provides a vector comprising a nucleic acid molecule of the invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like. The vectors or nucleic acid molecules of the invention can be used to transform or transfect host cells. Therefore, in a further aspect, the present invention provides a host cell comprising, or transformed or transfected with, the nucleic acid molecule and/or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

Antibodies or fragments thereof provided by the present invention can be obtained by any method known in the art. For example, host cells provided herein are cultured under conditions that allow the host cells to express the heavy and light chains of the antibody. Optionally, the method further comprises the step of recovering the antibodies produced.

Antibodies or fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be included in compositions, more particularly in pharmaceutical compositions such as pharmaceutical preparations, so as to be used for various purposes according to actual needs. Therefore, in yet another aspect, the present invention also provides a composition, such as a pharmaceutical composition, said composition comprising the antibody or fragment thereof, nucleic acid molecule, vector and/or host cell of the present invention, and optionally Pharmaceutically acceptable excipients.

Experiments have proved that the anti-CD3 antibody provided by the present invention has a reduced affinity to the antigen CD3, and after the heavy chain variable region and light chain variable region contained in it are constructed together with antibodies against other antigens to form a bispecific antibody, T cells can still be effectively recruited at the tumor cell site to achieve a good therapeutic effect. Therefore, the antibodies or fragments thereof (eg, functional regions) can be used to construct multispecific, especially bispecific antibodies, which function to bind CD3 on the surface of T cells to effectively recruit T cells without overstimulating T cells.

Therefore, in another aspect, the present invention also provides the anti-CD3 antibody or fragment thereof, nucleic acid molecule, vector, host cell and/or the use of the host cell in the preparation of a reagent for recruiting and activating T cells.

Correspondingly, in another aspect, the present invention provides the use of the combination of polypeptide 1 and polypeptide 2 in the preparation of a multispecific antibody construct, or in the preparation of a reagent for recruiting and activating T cells. Wherein, the polypeptide 1 comprises the amino acid sequence (TYAMN) shown in SEQ ID NO:22, the amino acid sequence (RIRSKYNNYATYYADSVKD) shown in SEQ ID NO:23, the amino acid sequence (HGNFGNSYVSYFAY) shown in SEQ ID NO:24; and , the polypeptide 2 comprises the amino acid sequence shown in SEQ ID NO: 25 (RSSTGAVTTSNYAN), the amino acid sequence shown in SEQ ID NO: 26 (GTNKRAP), the amino acid sequence shown in SEQ ID NO: 27 (ALWYSNLWV).

Preferably, said polypeptide 1 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with said amino acid sequence, said polypeptide 2 comprises the amino acid sequence shown in SEQ ID NO: 8 or An amino acid sequence having at least 75% identity with said amino acid sequence; preferably, in said multispecific antibody construct, said polypeptide 1 and polypeptide 2 serve as the heavy chain variable region and light chain variable region in the antibody construct, respectively chain variable regions to form the binding domain that binds CD3.

Preferably, the CD3 is CD3 on the surface of human T cells.

Preferably, said multispecific antibody construct is a bispecific antibody construct. Preferably, the multispecific antibody construct further comprises domains for binding to other antigens; further preferably, the other antigens are tumor-associated antigens, such as GPRC5D, Her2, HER3, BCMA, CLDN18, TROP2, PDL1, DLL3, CD25, EGFR, CCR8, CXCR4, CD123, Ep-CAM, CD19, CD20, GPC-3, PSMA, CD371 or VEGFR2.

Also correspondingly, in another aspect, the invention provides a multispecific antibody construct comprising a first binding domain that binds a first antigen and comprising a second binding domain that binds a second antigen. domain, wherein the first antigen is a tumor-associated antigen, and the second antigen is CD3.

Preferably, the first antigen is GPRC5D, Her2, HER3, BCMA, CLDN18, TROP2, PDL1, DLL3, CD25, EGFR, CCR8, CXCR4, CD123, Ep-CAM, CD19, CD20, GPC-3, PSMA, CD371 or VEGFR2. According to a specific embodiment of the present invention, the tumor-associated antigen is a group 5 D member of the G protein-coupled receptor family class C (GPRC5D).

Preferably, the second antigen is CD3 on the surface of human T cells.

Preferably, in the multispecific antibody construct provided by the present invention, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable regions (VL) comprising the following CDR combinations (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): comprising those shown in SEQ ID NO: 22 HCDR-1 comprising the amino acid sequence (TYAMN), HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:23 (RIRSKYNNYATYYADSVKD), HCDR-3 comprising the amino acid sequence (HGNFGNSYVSYFAY) shown in SEQ ID NO:24; and , LCDR-1 comprising the amino acid sequence (RSSTGAVTTSNYAN) shown in SEQ ID NO:25, LCDR-2 comprising the amino acid sequence (GTNKRAP) shown in SEQ ID NO:26, comprising the amino acid sequence shown in SEQ ID NO:27 (ALWYSNLWV) LCDR-3.

Further preferably, in said second binding domain, said heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/ Alternatively, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence at least 75% identical to said amino acid sequence.

According to a specific embodiment of the present invention, the multispecific antibody is a bispecific antibody.

Preferably, in the bispecific antibody construct provided by the present invention, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprising a combination of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR1, LCDR-2, LCDR-3):

(1) HCDR-1 comprising the amino acid sequence (SDYAWN) shown in SEQ ID NO:28, HCDR-2 comprising the amino acid sequence (YISYSGSATYSPSLKS) shown in SEQ ID NO:29, comprising HCDR-2 shown in SEQ ID NO:30 HCDR-3 of the amino acid sequence (GGIAGRGRWGAMDY); and, LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31, LCDR-2 comprising the amino acid sequence (SASYRDS) shown in SEQ ID NO:32, LCDR-3 comprising the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO:33;

(2) HCDR-1 comprising the amino acid sequence (SYTIQ) shown in SEQ ID NO:34, HCDR-2 comprising the amino acid sequence (YIIPSSGYTNYNQKFKD) shown in SEQ ID NO:35, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:36 HCDR-3 of the amino acid sequence (NYGNWGFTY); and, LCDR-1 comprising the amino acid sequence (KASQNVGSAVT) shown in SEQ ID NO:37, LCDR-2 comprising the amino acid sequence (SASNRYT) shown in SEQ ID NO:38, LCDR-3 comprising the amino acid sequence (QQYSNYPLT) shown in SEQ ID NO:39;

(3) HCDR-1 comprising the amino acid sequence (YYVMH) shown in SEQ ID NO:40, HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:41, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:42 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:43, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:44, LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:45; and

(4) HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:46, HCDR-2 comprising the amino acid sequence (YINPYNAGTKYNEKFKG) shown in SEQ ID NO:47, comprising HCDR-2 shown in SEQ ID NO:42 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:43, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:44, comprising the amino acid sequence shown in SEQ ID NO:44 LCDR-3 of the amino acid sequence (LQYATFPYT) of ID NO:45.

Further preferably, in the first binding domain, the heavy chain variable region and the light chain variable region comprise a combination of sequences selected from:

(1) the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:10 or the amino acid sequence shown in SEQ ID NO:10 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 10;

(2) the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 12;

(3) the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 13; and, the amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 14 an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 14; and

(4) the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 16.

Further, the bispecific antibody construct provided by the present invention comprises four polypeptide chains:

1) heavy chain 1, said heavy chain 1 comprising domains arranged according to VH-CH1-CH2-CH3 from the N-terminus to the C-terminus;

2) heavy chain 2, said heavy chain 2 comprising domains arranged according to VL-CH1-CH2-CH3 from N-terminus to C-terminus;

3) light chain 1 comprising domains arranged according to VL-CL from the N-terminus to the C-terminus;

4) light chain 2 comprising domains arranged according to VH-CL from N-terminus to C-terminus;

Wherein, the pairing of VH and VL contained in heavy chain 1 and light chain 1 forms the first binding domain that binds tumor-associated antigen; the pairing of VL and VH contained in heavy chain 2 and light chain 2 forms the first binding domain that binds to CD3. Second binding domain. The schematic diagram of the structure of the bispecific antibody construct provided by the present invention is shown in FIG. 1 .

According to a specific embodiment of the present invention, in the bispecific antibody construct provided by the present invention, the CH1-CH2-CH3 domain in the heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 19 or the amino acid sequence An amino acid sequence having at least 75% identity; the CH1-CH2-CH3 domain in heavy chain 2 comprising the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity to said amino acid sequence; light The CL domain in chain 1 comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence at least 75% identical to said amino acid sequence; the CL domain in light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to said amino acid sequence.

Compared with the prior art, the present invention provides a novel anti-CD3 antibody, which is obtained by humanizing the sequences of known mouse antibodies. Experiments have shown that, compared with the mouse antibody before transformation, the anti-CD3 antibody provided by the invention has a reduced affinity to the antigen CD3; After bispecific antibodies are constructed with antibodies against other antigens (such as tumor-associated antigens), they can still effectively recruit T cells at tumor cell sites and achieve good therapeutic effects.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 is a schematic diagram of the structure of an anti-human GPRC5D×CD3 bispecific antibody.

Figure 2 shows the detection results of the affinity between anti-CD3 antibody and CD3, wherein 2-1: GPRC5D×CD3 JNJ; 2-2: SP34V1 (hz); 2-3: SP34 (chi).

Figure 3 shows the affinity test results of anti-GPRC5D antibody and GPRC5D, wherein 3-1: GPRC5D×CD3 JNJ; 3-2: 9D7 (hz); 3-3: 26D1 (hz); 3-4: 24F5 (hz); 3-5: 6E97 (hz).

Figure 4 shows the detection results of bispecific antibody-mediated T cell killing, wherein 4-1: co-culture experiment 1; 4-2: co-culture experiment 2; 4-3: co-culture experiment 3.

Detailed ways

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only for illustrating the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

Example 1 Construction of control bispecific antibody

The anti-human GPRC5D×CD3 bispecific antibody was constructed by using the GPRC5D-targeting domain and the CD3-targeting domain of Johnson & Johnson's Talquetamab (JNJ-64407564). The structural diagram of the bispecific antibody is shown in Figure 1, in which the structural domains contained in each chain are as follows from the N-terminal to the C-terminal: heavy chain 1: VH-CH1-CH2-CH3

>VH(SEQ ID NO:1)

>CH1-CH2-CH3 (SEQ ID NO:19)

Heavy chain 2: VL-CH1-CH2-CH3

>VL(SEQ ID NO:2)

>CH1-CH2-CH3 (SEQ ID NO:20)

Light chain 1: VL-CL

>VL(SEQ ID NO:3)

>CL(SEQ ID NO:18)

Light chain 2: VH-CL

>VH(SEQ ID NO:4)

>CL(SEQ ID NO:21)

For the above four polypeptide chains, design primers, synthesize corresponding coding genes, connect them into eukaryotic expression plasmids, and co-transfect HEK293F cells with the obtained four recombinant expression plasmids. After 5-7 days of cell culture, harvest the supernatant that secretes antibodies solution; bispecific antibodies were purified from cell culture supernatants using MabSelectSure affinity chromatography and SP cation exchange chromatography. The obtained bispecific antibody was named GPRC5D×CD3 JNJ.

Example 2 Humanization of known mouse antibodies

The heavy chain variable region sequence (SEQ ID NO.10 in US10066015B2) and the light chain variable region sequence (SEQ ID NO.5 in US10066015B2) of the following mouse antibody were obtained from the patent US10066015B2.

>VH(SEQ ID NO:5)

>VL(SEQ ID NO:6)

The following human germline sequences were selected as templates for the heavy chain and light chain: IGHV3-11 and IGKV1-39. Homology modeling was performed on the A1 antibody, and the structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the A1 antibody was finally obtained.

By comparing and analyzing the predicted Fab structure and heavy chain and IGHV3-11 sequence, except for the CDR region and 37V and 76D in the VH, which are original mouse amino acids, all other mouse amino acids are replaced with the corresponding IGHV3-11 Template human amino acids.

By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, except for the CDR region and 39V, 49A, 52G, 69L, 72D, and 74A in the VL, the original mouse amino acids are retained. All substitutions were made with corresponding IGKV1-39 template human amino acids. The obtained humanized sequence is as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, which are obtained according to the KABAT definition method):

>VH humanized sequence (VH/HCDR-1/HCDR-2/HCDR-3: SEQ ID NO:7/22/23/24)

>VL humanized sequence (VL/LCDR-1/LCDR-2/LCDR-3: SEQ ID NO:8/25/26/27)

The sequence shown in SEQ ID NO: 17 was used as the heavy chain constant region, and the sequence shown in SEQ ID NO: 18 was used as the light chain constant region. For the above humanized sequences, primers were redesigned to synthesize the corresponding humanized antibody heavy chain and The coding gene of the light chain is connected into the eukaryotic expression vector, the recombinant vector is transformed into Escherichia coli competent cells, cultivated overnight at 37°C, and single-clonal strains are sequenced and identified. The strain with the correct sequence was selected to obtain the recombinant vector, which was transfected into mammalian expression cell 293F, and cultured for 7 days at 37°C and 5% CO2. The supernatant was collected and purified to obtain an antibody containing the above humanized sequence, which was named SP34V1 (hz); similarly, a chimeric antibody was obtained based on the heavy and light chain variable region sequences of the above murine antibody in the patent US10066015B2, named SP34V1 (hz). It is SP34(chi).

>CH1-CH3 heavy chain constant region (SEQ ID NO: 17)

>CL1 light chain constant region (SEQ ID NO: 18)

The humanized antibody, chimeric antibody and bispecific antibody GPRC5D×CD3 JNJ were prepared into a solution with a concentration of 10 μg/mL in PBS buffer, and the antibody was captured using an AHC chip according to the instructions of Fortebio. Let the human CD3 protein prepared in advance (Kaijia Biology, Cat. No. GPR-HM05P) (the highest concentration of protein is 200nM, 2-fold dilution, 6 gradients) flow through the chip, the specific conditions are: flow rate 30μl/min; antigen-antibody binding time 200 seconds, dissociation time 500 seconds. The measured results were fitted with the instrument-specific supporting software to analyze the affinity of the antibody to the antigen. The results are shown in Table 1 and 2-1 to 2-3 in Fig. 2 .

Table 1. Affinity test results of antibody and CD3 protein

Antibody KD(M) kon(1/Ms) kdis(1/s) GPRC5D×CD3 JNJ 6.51E-09 4.41E+05 2.88E-03 SP34V1(hz) 1.80E-08 4.42E+05 7.95E-03 SP34(chi) 5.46E-09 5.01E+05 2.74E-03

Example 3 The acquisition and detection of anti-human GPRC5D antibody

Cells expressing human GPRC5D (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. (kyinno company), Cat. No. KC-1583) were prepared into a cell suspension in PBS containing 2% FBS, and used as an antigen to immunize mice. The mouse antibody with high affinity to the antigen human GPRC5D was obtained by screening hybridoma cells, and then the heavy and light chain variable regions of the mouse antibody were humanized to obtain humanized VH and VL sequences.

According to Example 2, the sequence shown in SEQ ID NO: 17 is also used as the heavy chain constant region, and the sequence shown in SEQ ID NO: 18 is used as the light chain constant region, and humanized VH and VL sequences are used to obtain 4 strains The humanized antibodies against human GPRC5D are named 9D7 (hz), 26D1 (hz), 24F5 (hz), and 6E97 (hz) respectively, and the sequences of their respective heavy chain and light chain variable regions are as follows:

(1)9D7(hz):

>9D7-H humanized sequence (VH/HCDR-1/HCDR-2/HCDR-3: SEQ ID NO:9/28/29/30)

>9D7-L humanized sequence (VL/LCDR-1/LCDR-2/LCDR-3: SEQ ID NO:10/31/32/33)

(2) 26D1(hz):

>26D1-H humanized sequence (VH/HCDR-1/HCDR-2/HCDR-3: SEQ ID NO:11/34/35/36)

>26D1-L humanized sequence (VL/LCDR-1/LCDR-2/LCDR-3: SEQ ID NO:12/37/38/39)

(3) 24F5(hz):

>24F5-H humanized sequence (VH/HCDR-1/HCDR-2/HCDR-3: SEQ ID NO:13/40/41/42)

>24F5-L humanized sequence (VL/LCDR-1/LCDR-2/LCDR-3: SEQ ID NO:14/43/44/45)

(4) 6E97(hz):

>6E97-H humanized sequence (VH/HCDR-1/HCDR-2/HCDR-3: SEQ ID NO:15/46/47/42)

>6E97-L humanized sequence (VL/LCDR-1/LCDR-2/LCDR-3: SEQ ID NO:16/43/44/45)

The antibody was prepared into a solution with a concentration of 10 μg/mL using PBS buffer, and the AHC chip was used to capture the antibody according to the instructions of Fortebio. Let the human GPRC5D protein prepared in advance (Kaijia Biology, Cat. No. GPR-HM05P) (protein according to the highest concentration of 200nM, 2-fold dilution, 6 gradients) flow through the chip, the specific conditions are: flow rate 30μl/min; antigen-antibody binding time 200 seconds, dissociation time 500 seconds. The measured results were fitted with the instrument-specific supporting software to analyze the affinity of the antibody to the antigen. The results are shown in Table 2 and 3-1 to 3-5 in Fig. 3 .

Table 2. Affinity test results of humanized antibody and GPRC5D protein

Antibody KD(M) kon(1/Ms) kdis(1/s) GPRC5D×CD3 JNJ 9.47E-10 2.18E+04 2.06E-05 9D7(hz) <1.0E-12 2.26E+04 <1.0E-07 26D1(hz) 1.70E-09 1.85E+04 3.15E-05 24F5(hz) 1.36E-09 3.99E+04 5.42E-05 6E97(hz) 4.58E-10 4.55E+04 2.08E-05

Example 4 Construction of bispecific antibody

As described in Example 1, a bispecific antibody was constructed using the VH and VL of the anti-CD3 antibody and the VH and VL of the anti-GPRC5D antibody provided by the present invention. Compared with the control bispecific antibody GPRC5D×CD3 JNJ, the only difference is that the VH in heavy chain 1 and the VL in light chain 1 are replaced by humanized antibodies 9D7(hz), 26D1(hz), 24F5(hz) and 6E97, respectively (hz) VH and VL, the VL in the heavy chain 2 and the VH in the light chain 2 were replaced with the VH and VL of the humanized antibody SP34V1 (hz). The obtained bispecific antibodies were named as GPRC5D×CD3 9D7(hz), GPRC5D×CD3 26D1(hz), GPRC5D×CD3 24F5(hz), and GPRC5D×CD3 26D1(hz).

Example 5 GPRC5D and CD3 double antibody-mediated T cell killing experiment

PBMC and CD3/CD28 magnetic beads were mixed in RPMI1640 containing 10 ng/ml IL-2 and 10% FBS at a quantity ratio of 2:1, cultured and expanded for 7 days to activate T cells.

HiBiT Extracellular Reagent(Promega，货号N2420)检测上清中死亡细胞释放的LDHA-HIBIT，由此检测杀伤细胞比例。本实验针对2个抗体各设置了2组对照：加入293T LDHA-Hibit-GPRC5D细胞和抗体，但不加T细胞；加入293T LDHA-Hibit细胞(kyinno公司)、T细胞和抗体，以排除非抗体介导的杀伤和抗体介导的非T细胞杀伤。结果见图4中的4-1。 Co-culture experiment 1: 293T LDHA-Hibit-GPRC5D cells (kyinno, product number KC-2151) were plated (96-well plate) one day in advance, and then 10,000 of the above T cells were added to each well, and different concentrations of positive control antibody GPRC5D were added ×CD3 JNJ and the bispecific antibody of the present invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001 μg/ml) were co-cultured in 18 RPMI1640 containing 10ng/ml IL-2 and 10% FBS Hour. use

HiBiT Extracellular Reagent (Promega, Cat. No. N2420) detects LDHA-HIBIT released from dead cells in the supernatant, thereby detecting the ratio of killer cells. In this experiment, two groups of controls were set up for each of the two antibodies: 293T LDHA-Hibit-GPRC5D cells and antibodies were added, but T cells were not added; 293T LDHA-Hibit cells (kyinno company), T cells and antibodies were added to exclude non-antibody mediated killing and antibody-mediated non-T cell killing. The results are shown in 4-1 in Fig. 4.

Co-culture experiment 2: NCI-H929 (expressing GPRC5D) and K562 cells (not expressing GPRC5D) were stained with CFSE and named CFSE-H929 and CFSE-K562, respectively. Add 10,000 activated T cells and 3,000 CFSE-H929 to each well of a u-bottomed 96-well plate, and add different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibody of the present invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001 μg/ml), co-cultivated in RPMI1640 containing 10 ng/ml IL-2 and 10% FBS for 18 hours. Then, it was stained with 7-AAD and detected by flow cytometry, and the ratio of CFSE+7-AAD+ cells in CSFE+ cells was counted as the killing ratio of cells. In this experiment, two groups of controls were set up for each of the two antibodies: CFSE-H929 cells and antibodies were added, but T cells were not added; CFSE-K562, T cells and antibodies were added to exclude non-antibody-mediated killing and antibody-mediated killing. Non-T cell killing. The results are shown in 4-2 in Fig. 4.

Co-culture experiment 3: wild-type NCI-H929 (kyinno company, product number KC-0629) and GPRC5D knockout NCI-H929 cells (kyinno company, product number KC-2203) were stained with CFSE, named CFSE-H929 and CFSE respectively -H929GPRC5DKO. Add 10,000 activated T cells, 3,000 CFSE-H929, and different concentrations of positive control antibody GPRC5D×CD3 JNJ and the bispecific antibody of the present invention (1, 0.1, 0.01 , 0.001, 0.0001, 0.00001, 0.000001, 0.0000001 μg/ml), co-cultured in RPMI1640 containing 10 ng/ml IL-2 and 10% FBS for 18 hours. Then, it was stained with 7-AAD and detected by flow cytometry, and the ratio of CFSE+7-AAD+ cells in CSFE+ cells was counted as the killing ratio of cells. In this experiment, two groups of controls were set up for each of the four antibodies: CFSE-H929 cells and antibodies were added, but T cells were not added; CFSE-H929GPRC5DKO, T cells and antibodies were added to exclude non-antibody-mediated killing and antibody-mediated killing. Non-T cell killing. The results are shown in 4-3 in Fig. 4.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (15)

An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region ( VL) comprises the following CDR combinations (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): HCDR-1 comprising the amino acid sequence shown in SEQ ID NO: 22, comprising the HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:23, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:24; and LCDR-1 comprising the amino acid sequence shown in SEQ ID NO:25 comprising the amino acid sequence shown in SEQ ID NO:25 LCDR-2 of the amino acid sequence of ID NO:26, LCDR-3 comprising the amino acid sequence shown in SEQ ID NO:27. The antibody or fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence; And/or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to said amino acid sequence. The antibody or fragment thereof according to claim 1 or 2, wherein the antibody or fragment thereof is an anti-CD3 antibody or fragment thereof; Preferably, the antibody is in any form such as mouse anti-antibody, rabbit anti-antibody, monoclonal antibody, chimeric antibody, partially or fully humanized antibody; or, the fragment is a half-antibody or an antigen-binding fragment of an antibody or half-antibody , for example scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; More preferably, the antibody is IgG. A nucleic acid molecule comprising a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a heavy chain or Nucleotide sequence of the light chain. A vector comprising the nucleic acid molecule of claim 4. A host cell comprising the nucleic acid molecule of claim 4 and/or the vector of claim 5. A composition comprising the antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, and/or the host of claim 6 cell; Preferably, the composition is a pharmaceutical composition, which also includes optional pharmaceutically acceptable auxiliary materials. The antibody or fragment thereof of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, and/or the nucleic acid molecule of claim 7 Use of the composition of the invention in the preparation of a reagent for recruiting and activating T cells. Use of a combination of polypeptide 1 and polypeptide 2 in the preparation of a multispecific antibody construct, wherein the polypeptide 1 comprises the amino acid sequence shown in SEQ ID NO: 22, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in SEQ ID NO: The amino acid sequence of ID NO:24; And, said polypeptide 2 comprises the amino acid sequence shown in SEQ ID NO:25, the amino acid sequence shown in SEQ ID NO:26, the amino acid sequence shown in SEQ ID NO:27. Use of a combination of polypeptide 1 and polypeptide 2 in the preparation of a reagent for recruiting T cells, wherein the polypeptide 1 comprises the amino acid sequence shown in SEQ ID NO: 22, the amino acid sequence shown in SEQ ID NO: 23, the amino acid sequence shown in The amino acid sequence of SEQ ID NO:24; And, the polypeptide 2 comprises the amino acid sequence shown in SEQ ID NO:25, the amino acid sequence shown in SEQ ID NO:26, the amino acid sequence shown in SEQ ID NO:27. The use according to claim 9 or 10, wherein said polypeptide 1 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with said amino acid sequence, said polypeptide 2 comprising the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to said amino acid sequence; Preferably, in the multispecific antibody construct, the polypeptide 1 and polypeptide 2 are combined as the heavy chain variable region and the light chain variable region in the antibody construct respectively to form a binding domain that binds to CD3. The use according to any one of claims 9 to 11, wherein the multispecific antibody construct is a bispecific antibody construct; Preferably, the multispecific antibody construct further comprises domains for binding to other antigens; further preferably, the other antigens are tumor-associated antigens, such as GPRC5D, Her2, HER3, BCMA, CLDN18, TROP2, PDL1, DLL3, CD25, EGFR, CCR8, CXCR4, CD123, Ep-CAM, CD19, CD20, GPC-3, PSMA, CD371 or VEGFR2. A multispecific antibody construct comprising a first binding domain that binds a first antigen and comprising a second binding domain that binds a second antigen, wherein the first antigen is a tumor Related antigen, the second antigen is CD3; the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VH) The chain variable region (VL) comprises the following combination of CDRs (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): HCDR comprising the amino acid sequence shown in SEQ ID NO:22 -1. HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:23, HCDR-3 comprising the amino acid sequence shown in SEQ ID NO:24; and, an LCDR comprising the amino acid sequence shown in SEQ ID NO:25 -1. LCDR-2 comprising the amino acid sequence shown in SEQ ID NO:26, LCDR-3 comprising the amino acid sequence shown in SEQ ID NO:27. The antibody construct according to claim 13, wherein the first antigen is GPRC5D, Her2, HER3, BCMA, CLDN18, TROP2, PDL1, DLL3, CD25, EGFR, CCR8, CXCR4, CD123, Ep-CAM , CD19, CD20, GPC-3, PSMA, CD371 or VEGFR2; Preferably, the second antigen is CD3 on the surface of human T cells; Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or, the light chain variable region comprises the amino acid sequence shown in The amino acid sequence at SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to said amino acid sequence. The antibody construct according to claim 13 or 14, wherein the multispecific antibody construct is a bispecific antibody construct; Preferably, the bispecific antibody construct comprises four polypeptide chains: 1) heavy chain 1, said heavy chain 1 comprising domains arranged according to VH-CH1-CH2-CH3 from the N-terminus to the C-terminus; 2) heavy chain 2, said heavy chain 2 comprising domains arranged according to VL-CH1-CH2-CH3 from N-terminus to C-terminus; 3) light chain 1 comprising domains arranged according to VL-CL from the N-terminus to the C-terminus; 4) light chain 2 comprising domains arranged according to VH-CL from N-terminus to C-terminus; Wherein, the pairing of VH and VL contained in heavy chain 1 and light chain 1 forms the first binding domain that binds tumor-associated antigen; the pairing of VL and VH contained in heavy chain 2 and light chain 2 forms the first binding domain that binds to CD3. Second binding domain.

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