Humanized chimeric antigen receptor targeting B cell receptor of chronic lymphocytic leukemia and application thereof

Abstract

The present invention provides a humanized chimeric antigen receptor (CAR) that targets the B cell receptor (BCR) of a CLL cell characterized by an R110 mutated immunoglobulin lambda variant 3-21 (IGLV3-21R110). The invention also provides a nucleic acid sequence for coding the CAR, a vector containing the nucleic acid sequence, a pharmaceutical composition and a kit with instructions for use.

Description

Technical Field

[0001] The present invention provides humanized chimeric antigen receptors (CARs) for the treatment of chronic lymphocytic leukemia (CLL). These CARs target the B cell receptor (BCR) of CLL cells, which are characterized by the immunoglobulin lambda variant 3-21 (IGLV3-21) with R110 mutation. R110 ).

[0002] The present invention also provides a nucleic acid sequence encoding the aforementioned CAR, a vector comprising the nucleic acid sequence, a cell expressing the nucleic acid sequence, a pharmaceutical composition, and a kit with instructions for use. Background Art

[0003] Adoptive immunotherapy using chimeric antigen receptor (CAR) redirected immune effector cells is becoming a promising approach to treat leukemia and lymphoma derived from malignant transformation of B-lineage cells. Since the approval of CAR-T cell therapy involving the use of CAR directed against the common B-cell antigen "CD19", the response rate, long-term outcome and quality of life of patients with certain types of B-cell type leukemia and lymphoma (such as acute lymphoblastic leukemia, diffuse large B-cell lymphoma and mantle cell lymphoma) have been significantly improved (e.g., Maude SL, Frey N, Shaw Pa, et al. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia; N Engl J Med 2014; 371: 1507-15017).

[0004] The most common type of leukemia in Western countries is chronic lymphocytic leukemia, which usually occurs in elderly patients. The risk of men developing CLL is twice that of women (Kipps TJ, Stevenson FK, Wu CJ, Croce CM, Packham G, Wierda WG, et al. Chronic lymphocytic leukemia. Nat Rev Dis Primers (2017) 3: 1-12). CLL is a malignant tumor derived from heterogeneous B lymphocytes. The malignant tumor is caused by the clonal proliferation of CD5-positive B lymphocyte subsets that gradually accumulate in the bone marrow, lymph nodes, peripheral blood and spleen (Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med. 1995; 333: 1052-1057).

[0005] As reviewed by Burger and Chiorazzi, clinical and biological evidence indicates that the BCR is one of the major factors in the clonal selection and survival of CLL cells (Burger JA, Chiorazzi NB cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol 2013; 34: 592-601).

[0006] BCR is a multi-protein structure composed of non-covalently bound antigen binding subunits and signal transduction subunits. The antigen binding subunit is composed of a membrane immunoglobulin comprising two identical heavy chains and two identical light chains, with one constant domain in each light chain and three constant domains in each heavy chain. Each heavy chain is combined with a light chain to form an antigen binding site. Each light chain and each heavy chain contain variable domains that form antigen binding sites. The immunoglobulin genes encoded in Igh, Igl and Igk loci contain a large number of V (variable), D (diversity) and J (joining) gene segments located upstream of one or more constant exons. In developing B cells, the gene rearrangement of immunoglobulins randomly assembles V, D and J gene segments to form complete V exons in the Igh locus and V and J gene segments in the Igk or Igl locus. Through combinatorial joining of gene segments, junctional diversity, and random heavy and light chain pairing, each individual B cell progenitor generates its own and nearly unique antigen-binding subunit, whose antigen-binding affinity can be further refined by somatic hypermutation (SHM).

[0007] The signal transduction part of BCR is composed of a disulfide-linked heterodimer of Igα and Igβ (CD79a / CD79b) proteins. In their cytoplasmic tails, Igα and Igβ each contain a separate immunoreceptor tyrosine activation motif (ITAM), which initiates signal transduction once antigen binding is followed by BCR aggregation (Flaswinkel, H., Reth, M., 1994. Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor. EMBO J. 13, 83–89).

[0008] Antigen binding rapidly activates Src family kinase Lyn, leading to Igα / Igβ phosphorylation. This triggers the formation of a large signal complex on the cytoplasmic side of the membrane, which is composed of BCR, various tyrosine kinases, adapter proteins and signal enzymes. Proximal BCR signaling is mediated by protein tyrosine kinase Syk (spleen tyrosine kinase), which is recruited to the phosphorylated ITAM of Igα and Igβ via Syk and adapter protein SLP65 and its downstream signal enzyme Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2) combination, resulting in the propagation of the signal. The signal of the signal complex is activated downstream pathways, including calcium mobilization, phosphoinositide 3-kinase (PI3K), nuclear factor-κB (NF-κB), activated T cell nuclear factor (NF-AT), mitogen-activated protein kinase (MAPK) and rat sarcoma (RAS) signaling pathway (Burger JA and Chiorazzi N, 2013, supra).

[0009] Chronic activation of mature B cells through B cell receptors has been shown to be a key process in the formation and development of CLL (Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham GB-cell receptor signaling in chronic lymphocytic leukemia Blood. 2011; 118: 4313-4320). This is also consistent with a study using the EBV protein LMP2A as a constitutively active BCR substitute, which showed that the development of the mouse B1 subpopulation depends on strong and persistent BCR stimulation (Casola S, Otipoby KI, Alimzhanov M, et al. B cell receptor signal strength determines B cell fate. Nat Immunol 2004; 5: 317–27). In addition, the interaction of two adjacent BCRs on a cell leads to antigen-independent, autonomous signaling of primary CLL B cells, which has been identified as a key driver of CLL development, resulting in increased tyrosine phosphorylation of BCR proximal signaling molecules, leading to cyclic signaling and Ca 2+ Mobilization is increased (Dühren-von Minden, et al. Chronic lymphocyticleukemia is driven by antigen-independent cell-autonomous signalling. Nature. 2012; 489: 309-313).

[0010] There is ample evidence that protein kinase Syk is constitutively phosphorylated by sustained BCR signaling, and several studies have revealed that other key molecules in the downstream signaling pathways involved in BCR in normal B cells, such as PKC, phosphoinositide 3-kinase, and mitogen-activated protein kinase p38, are also constitutively activated in B-CLL cells, resulting in deregulation of the activity or expression of multiple pro-survival molecules and downstream pathways (Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Sica S, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1and induces apoptosis in chronic lymphocyticleukemia B cells. Leukemia 2009; 23: 686–697. Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, et al. Constitutively activatedphosphatidylinositol-3kinase(PI-3K) is involved in the defect of apoptosis inB-CLL:association with protein kinase C delta.Blood 2002;100:3741–3748.PlateJM.PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells bypreventing caspase 8activation.Leuk Lymphoma 2004;45:1519–1529. Sainz-Perez A, Gary-Gouy H, Portier A, Davi F, Merle-Beral H, Galanaud P, et al. High Mda-7expression promotes malignant cell survival and p38 MAP kinase activation inchronic lymphocytic leukemia. Leukemia 2006; 20:498–504.).

[0011] Constitutively activated signaling pathways, such as NF-kB or PI3K / AKT, have been shown to lead to the transcription and overexpression of key anti-apoptotic proteins, particularly B-cell lymphoma 2 (Bcl-2) and several members of the inhibitor of apoptosis protein (IAP) family (Loeder S, et al. A novel paradigm to trigger apoptosis in chronic lymphocytic leukemia. Cancer Res. 2009; 69: 8977-8986). It has been confirmed that Mcl-1, in addition to Bcl-2 itself, is a key factor in impaired apoptosis in CLL cells, and BCR signaling has been reported to upregulate Mcl-1 expression through the PI3K / AKT pathway (Petlickovski A, Laurenti L, Li X, Marietti S, Chiusolo P, Sica S, Leone G, Efremov DG. Sustained signalling through the B-cell receptor induces Mcl-1and promotes survival of chronic lymphocytic leukemia B cells. Blood. 2005; 105: 4820-4827).

[0012] It is well established that different aspects of the BCR are used to identify the major CLL disease subtypes. For example, the level of somatic hyperfrequency mutations within the variable region of the BCR immunoglobulin heavy chain (IGHV) has been used as a prognostic marker for decades. CLL patients with mutated IGHV genes (M-CLL) (i.e., showing less than 98% IGHV gene identity with their closest germline) generally have a slower disease progression than CLL patients with unmutated IGHV genes (U-CLL) with germline identity equal to or greater than 98%. However, exceptions to this rule have also been observed, in which the mutated IGHV gene status is not associated with the progression of a certain disease. For example, cases using the IGHV3-21 gene, although most of them express mutated BCRs, have one of the worst clinical outcomes. A different approach, but also determined by IGHV, has resulted in approximately 30% of CLL cases being classified into important subgroups with different prognoses, each with highly homogeneous biological characteristics, clinical manifestations, and outcomes. This classification is based on the observation that there are stereotyped BCRs carrying highly homologous heavy chain complementary determining region 3 (H-CDR3) sequences in mutated and unmutated cases. According to this approach, CLL cases characterized by mutated IGHV3-21 can be classified into the so-called subgroup #2 (Stamatopoulos K, Belessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenic implications and clinical correlations. Blood. 2007; 109(1): 259-270; Agathangelidis A., et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: A molecular classification with implications for targeted therapies. Blood. 2012; 119: 4467–4475).

[0013] It is noteworthy that the use of IGHV3-21 was consistently observed to be associated with the expression of immunoglobulin lambda variable region 3-21 chains and the addition of amino acid 110 in the light chain (IGLV3-21 R110 ) is associated with the substitution of glycine by arginine. R110The reason for the arginine 110 (R110) in BCRs is a single G>C substitution at the splice site between the immunoglobulin λ J and constant genes. It has been identified that the co-presence of R110 in one BCR with the germline-encoded lysine 16 (K16) and aspartates (D) 50 and 52 in the tyrosine-aspartate-serine-aspartate (YDSD) motif of the adjacent BCR enables BCR-BCR interactions, thereby triggering cell-autonomous signaling ( Fig.13 and Figure 14; Minici, C. et al., Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukemia, Nature Comm. 2017;8:15746).

[0014] During the epigenetic, genomic, and transcriptomic characterization of a large model cohort of CLL patients with a focus on the BCR light chain, it became apparent that approximately 60% of IGLV3-21 R110 Cases carried atypical BCRs, which emphasizes that subgroup #2 is only based on IGLV3-21 R110 A small subgroup of CLL characterized by (Stamatopoulos B, Smith T, Crompot E, et al. The Light Chain IgLV3-21 Defines a New Poor Prognostic Subgroup in Chronic Lymphocytic Leukemia: Results of a Multicenter Study. Clin Cancer Res. 2018; 24(20): 5048-5057. Nadeu F, Royo R, Clot G, et al. IGLV3-21 R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia within termediate epigenetics. Blood. 2021;137(21):2935–2946).

[0015] Of the four alleles of the IGLV3-21 gene identified in humans, alleles IGLV3-21*01 (IMGT / LIGM-DB, accession number X71966) and IGLV3-21*04 (IMGT / LIGM-DB, accession number AC279208) encode the prerequisites K16 and D50 and D52, the latter two of which are merged into a motif that, in most of the cases studied, encompasses the IGLV3-21 R110 Tyrosine at position 49 and serine at position 51 (exemplary IGLV3-21 R110 , see Figure 14). However, in IGLV3-21 R110 Functionally equivalent variations of this motif have also been observed in CLL patients, such as substitution of phenylalanine for tyrosine or threonine for serine (Nadeu, et al. 2021, supra). Interestingly, alleles IGLV3-21*01 and IGLV3-21*04 were clearly underrepresented in B cells from healthy donors, whereas in patients studied in different groups all IGLV3-21 genes could be assigned to either allele IGLV3-21*01 or allele IGLV3-21*04, suggesting that these alleles could be IGLV3-21 R110 Required for the development of relevant CLL mechanisms.

[0016] IGLV3-21 R110 The CLL subgroup, which has been proposed to be designated subgroup #2L, is associated with a very aggressive disease course. R110 The adverse outcome in CLL patients is independent of the IGHV mutation status or the nature of the heavy chain. R110 It is found in mutated CLL (such as subgroup #2 (see above)) and is associated with different heavy chains (such as IGHV1-18, IGHV3-53 or IGHV3-64) (Nadeu, et al. 2021, supra). Therefore, IGLV3-21 R110 We define a group of CLL that is neither restricted to the typical empirically defined epigenetic subgroups nor to the IGHV mutational status. Furthermore, the importance of R110 as a CLL driver mutation has been confirmed by site-specific mutagenesis experiments, which revealed that IGLV3-21 R110 Reversion to IGLV3-21G110 results in loss of BCR-autonomous signaling ability (Stamatopoulos B, Smith T, Crompot E, et al. 2018, supra).

[0017] Time to first treatment (TTFT) and overall survival (OS) are associated with the presence of IGLV3-21 R110 Studies on the association of BCR with IGLV3-21 have shown that R110 Compared with CLL patients, those expressing IGLV3-21 R110 The values ​​of patients with IGLV3-21 were significantly shorter, which emphasizes that R110 Positive patients require faster treatment (Nadeu F et al. 2021; ibid.).

[0018] However, since CARs targeting common B cell antigens (such as CD19) cannot distinguish between healthy and malignant B cells, patients will experience persistent B cell depletion as long as anti-CD19 CAR T cells are present. Due to severe hematological toxicity leading to immunosuppression, inability to form vaccine responses, high risk of infection, and off-target effects on the immune system, no CAR has been shown to be suitable for treating CLL (see, e.g., Porter DL, Levine BL, Kalos M, et al. Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia; N Engl J Med 2011; 365: 725-733).

[0019] Therefore, it remains to be seen whether a specific CAR can be used to treat IGLV3-21. R110 Positive CLL patients without the above side effects.

[0020] CAR is an artificially constructed chimeric transmembrane protein or polypeptide. The transmembrane domain anchors CAR in the cell membrane of immune cells and connects the extracellular domain including the antigen binding domain to the cytoplasmic domain that provides effector functions for immune cells. Immune effector cells engineered to express the CAR gene redirect cells to kill tumors expressing surface antigens targeted by the antigen binding domain of CAR. The characteristics of CAR include the ability to redirect the specificity and reactivity of immune cells to the selected target in an MHC-independent manner using the antigen binding properties of antibodies. The antigen binding domain is traditionally derived from monoclonal antibodies. WO 2019 / 008129 discloses a monoclonal antibody that can be used to remove CLL cells from blood samples as a candidate for a fusion protein with a T cell-specific activation domain to obtain CAR. However, no therapeutic effect of such CAR has been shown. In addition, the antibodies in WO 2019 / 008129 have been expressed as IgG antibodies in hybridoma cell lines derived from mouse hosts. The potential problem of using mouse monoclonal antibodies as the basis of CAR is that they can be recognized as foreign objects and can induce immune responses in patients. This immunogenicity can lead to poor persistence in patients, and responses such as human anti-mouse antibody (HAMA) responses can prevent repeated administration of CAR-exposed cells.

[0021] In light of the above, a therapeutic option, particularly a CAR that is selective for healthy B cells and does not cross-react, would improve the efficacy of IGLV3-21. R110 Treatments for CLL in HER2-positive patients, with reduced risk of toxicity and immunogenic responses, remain unknown but are needed. Summary of the invention

[0022] The present invention solves the above problems by providing the following aspects. In the first aspect, a novel humanized CAR comprises humanized IGLV3-21 R110 Binding domain, transmembrane domain and cytoplasmic domain, wherein the humanized IGLV3-21 R110The binding domain includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) includes a light chain complementary determining region 1 (L-CDR1) having an amino acid sequence of SEQ ID NO: 1, a light chain complementary determining region 2 (L-CDR2) having an amino acid sequence of SEQ ID NO: 2, and a light chain complementary determining region 3 (L-CDR3) having an amino acid sequence of SEQ ID NO: 3, and the heavy chain variable region (VH) includes a heavy chain complementary determining region 1 (H-CDR1) having an amino acid sequence of SEQ ID NO: 4, a heavy chain complementary determining region 2 (H-CDR2) having an amino acid sequence of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (H-CDR3) having an amino acid sequence of SEQ ID NO: 6.

[0023] This humanized CAR specifically binds to the IGLV3-21 R110 The IGLV3-21 recognizes the BCR and thus, unlike anti-CD19 CAR, mediates the killing of malignant B cells without causing off-target killing of healthy B cells in CLL patients. R110 This new CAR can also target “subgroup #2CLL”, which is characterized by the inclusion of IGHV3-21 / IGLV3-21 R110 Combined BCR, or including IGLV3-21 R110 any other disease or CLL.

[0024] In a second aspect, the present invention also relates to a polynucleotide encoding a humanized CAR of the present invention. Therefore, in a third aspect, the present invention also relates to a vector comprising the nucleic acid sequence of the present invention.

[0025] In a fourth aspect, the present invention relates to a cell expressing a humanized CAR according to the present invention.

[0026] In a fifth aspect, the present invention relates to a pharmaceutical composition according to the present invention.

[0027] In a sixth aspect, the present invention relates to providing a CAR, a polynucleotide, a vector, a cell, or a pharmaceutical composition according to the present invention for treating a disease, particularly IGLV3-21 R110 Similarly, the sixth aspect of the present invention relates to a method for treating a disease, in particular a method for treating IGLV3-21 R110 A method for treating CLL in a positive patient by administering a CAR, polynucleotide, vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof.

[0028] According to the sixth aspect, the CAR, polynucleotide, vector, cell or pharmaceutical composition can be administered in combination with another therapeutic agent. Specifically, the therapeutic agent can be Bruton's tyrosine kinase (BTK).

[0029] On the other hand, a kit is provided, which includes a CAR, a polynucleotide, a vector, a cell, or a pharmaceutical composition according to the present invention.

[0030] definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. However, the following references can provide general definitions of many of the terms used in the present invention for those of ordinary skill in the art to which the invention belongs, and can be cited and used as long as such definitions are consistent with the meanings commonly understood in the art. Such references include, but are not limited to: Singleton et al. Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al. The Dictionary of Cell & Molecular Biology ((3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, WB Saunders Company. One of ordinary skill in the art can consult any additional technical resources that provide definitions of the terms used herein, the meanings of which are generally understood in the art. For the purposes of the present invention, the following terms are further defined.

[0032] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

[0033] "Autonomously active" BCRs are a special type of permanently active BCR. While conventional activation is based on external antigens, the autonomously active BCRs are generated by their interaction with membrane structures on the same cell surface. For the clinical picture of CLL, autonomous activation-triggered interactions between BCRs adjacent to each other on the same cell surface have been shown (e.g., M. Dühren-von Minden et al.; Nature 2012).

[0034] IGLV3-21 R110 It is the light chain variable region of BCR, which is capable of BCR-BCR interaction to induce autonomous active BCR. R110 The characterised in that it has a sequence identity of greater than 80% with the sequence represented by SEQ ID NO: 41, wherein in any case, position 110 of the sequence is arginine instead of glycine.

[0035] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule, preferably composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), which are usually connected internally by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can include, for example, three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into: hypervariable regions, called complementarity determining regions (CDRs), and interspersed with more conserved regions, called framework regions (FRs), which provide suitable scaffolds for the CDRs.

[0036] As used herein, the term "complementarity determining region (CDR; e.g., CDR1, CDR2, and CDR3)" refers to the amino acid residues of the variable domain of a molecule capable of binding to an antigen, such as an antibody, antibody fragment, single-chain variable fragment (scFv), the presence of which is essential for antigen binding. Each variable domain typically has three CDR regions, defined as CDR1, CDR2, and CDR3. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Chothia and Lesk (J Mol Biol 196:901-917 (1987)), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). Each complementary determining region includes amino acid residues defined by IMGT. In some cases, the complementary determining region may also include amino acids from CDR regions defined according to Kabat and / or hypervariable loops defined according to the Chothia numbering system.

[0037] As used herein, the term "CAR" or "chimeric antigen receptor" is intended to refer to an artificial transmembrane protein that applies an antigen-triggered response to an immune cell. CAR engineered immune cells can be, for example, naive T cells, central memory T cells, effector memory T cells, or natural killer (NK) cells. CAR is generally composed of an extracellular domain and a cytoplasmic domain connected by a transmembrane domain. The extracellular domain includes at least an antigen binding domain.

[0038] Here, "antigen binding domain" is defined as the polypeptide portion of the CAR that recognizes the antigen. Specifically, the antigen binding domain of the present invention is a polypeptide having an antigen binding site including a complementary determining region (CDR). The antigen binding domain may include 6 CDRs and have antigen binding sites equivalent to the heavy chain variable region (VH) and light chain variable region (VL) of a classical antibody. The VH of a classical antibody is produced by the rearrangement of immunoglobulin genes of the V (variable), D (diversity) and J (joining) region gene fragments, while the VL is produced by the rearrangement of the V and J gene fragments. Each VH and VL generated in this way is typically composed of three CDRs and up to four FRs, arranged from the amino terminus to the carboxyl terminus, for example in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antigen binding domain is typically a single-chain variable fragment (scFv) derived from an antibody, but it can be based on other forms, including Fab, Fab', F(ab') 2 , VH and VL antibody chains (Fv fragments), full-length heavy and light chains, and linear antibodies.

[0039] An "antigen binding site" is typically found in one or more hypervariable regions, such as CDR1, CDR2, and / or CDR3 regions; however, the variable "framework" regions may also play an important role in antigen binding, such as by presenting the antigen binding site in an appropriate manner so that it binds the antigen.

[0040] As used herein, "humanized" CAR includes humanized antigen binding domains to reduce immunogenicity. The humanized form of non-human (e.g., mouse) antigen binding domains is a genetically engineered chimeric antigen binding domain, whose variable regions VL and VH include minimal sequences derived from non-human immunoglobulins. The variable region of an antigen binding domain can be one of the following: (i) CDR transplanted, wherein the CDR of the variable region is from a non-human source, and one or more frameworks of the variable region are human; (ii) wherein the amino acids in the framework region of the non-human variable region are partially exchanged for human amino acid sequences by genetic engineering, or (iii) derived from a non-human source (e.g., a transgenic mouse carrying a heterologous immune system), whose variable region is based on a human germline sequence. Therefore, a humanized antigen binding domain can be an antigen binding domain with some or all CDRs from non-human immunoglobulins and a variable region framework sequence of one or more frameworks (FR) from a human source. Similarly, humanized VL can have at least one, two or three CDRs from non-human immunoglobulins and variable region framework sequences from one, two, three or four frameworks (FR) of human origin. Similarly, humanized VH can have at least one, two or three CDRs from non-human immunoglobulins and variable region framework sequences from one, two, three or four frameworks (FR) of human origin. In some cases, the variable region framework residues of human origin can be replaced by corresponding non-human residues. In some cases, humanized variable regions can include separate residues that are neither found in human variable regions nor found in non-human immunoglobulin CDRs. If the antigen binding domain comprises at least a portion of an immunoglobulin constant region (Fc), the portion is typically a portion of a human immunoglobulin.

[0041] "Percent (%) sequence identity" is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the nucleic acid or amino acid residues in the reference polynucleotide sequence or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various means within the skill in the art, for example, using publicly available computer software such as BLAST (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403-10), BLAST-2, ALIGN, LALIGN or Megalign (DNASTAR) software, or immunogenetics Information DomainGapAlign tool (Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3D structure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, Tcell receptors, MHC, IgSF and MhcSF. Nucleic Acids Res. 2010; 38, D301-307). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0042] As used herein, a CAR that "specifically binds to," "is specific for," or "specifically recognizes" an antigen of interest, such as a tumor-associated polypeptide antigen target (here, IGLV3-21 R110 ), capable of distinguishing such antigens from one or more reference antigens. In its most general form, "specific binding", "specifically binding", "specific for" or "specifically recognizing" refers to the ability of the CAR to distinguish an antigen of interest from an unrelated antigen, such as determined by one of the following methods. Such methods include, but are not limited to, flow cytometry, Western blot, ELISA test, RIA test, ECL test, IRMA test, immunohistochemistry test and peptide scanning.

[0043] "Binding affinity" or "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" or "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The dissociation rate constant K D Usually based on the equilibrium binding constant (k a ) and the dissociation rate constant (k d ) is calculated from the ratio of D " is often used to describe the affinity between a molecule (such as an antibody) and its binding ligand (such as an antigen), that is, how tightly the ligand binds to a specific protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. The term "high affinity" refers to a molecule / antibody that has a binding affinity of less than or equal to 10 -9 The affinity (KD) of M (monovalent affinity) and IGLV3-21 R110Positive CLL BCR binding. Compared with other unrelated molecules, the molecule / antibody can have a substantially stronger affinity for the target antigen. Affinity can be measured by common methods known in the art, for example according to Example 1.

[0044] The "variants" of CAR contemplated by the present invention are those that retain their antigen binding domains for IGLV3-21. R110 A molecule with binding activity.

[0045] The term "cancer" refers to the physiological condition or disease in which cells divide uncontrollably, resulting in unregulated cell growth. A "tumor" includes one or more cancer cells. Specific implementation plan

[0046] First aspect of the present invention - CAR

[0047] The humanized CAR of the present invention specifically binds to the IGLV3-21 R110 CARs and their beneficial properties that enable therapeutic activity are described in more detail below.

[0048] According to a first aspect of the present invention, a humanized chimeric antigen receptor (CAR) is provided, which comprises a humanized anti-IGLV3-21 R110 Binding domain, transmembrane domain and cytoplasmic domain, wherein the humanized IGLV3-21 R110 The binding domain includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) includes a light chain complementary determining region 1 (L-CDR1) having an amino acid sequence of SEQ ID NO: 1, a light chain complementary determining region 2 (L-CDR2) having an amino acid sequence of SEQ ID NO: 2, and a light chain complementary determining region 3 (L-CDR3) having an amino acid sequence of SEQ ID NO: 3, and the heavy chain variable region (VH) includes a heavy chain complementary determining region 1 (H-CDR1) having an amino acid sequence of SEQ ID NO: 4, a heavy chain complementary determining region 2 (H-CDR2) having an amino acid sequence of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (H-CDR3) having an amino acid sequence of SEQ ID NO: 6.

[0049] Preferred embodiments of the first aspect of the invention are further characterized in more detail in Table 3 of the Examples section.

[0050] Therefore, a preferred embodiment of the first aspect of the present invention is a CAR, characterized in that it comprises any combination of a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12.

[0051] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H1" having a VL with the amino acid sequence of SEQ ID NO:7 and a VH with the amino acid sequence of SEQ ID NO:11 is a first more preferred embodiment.

[0052] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H2" having a VL with the amino acid sequence of SEQ ID NO: 7 and a VH with the amino acid sequence of SEQ ID NO: 12 is the second more preferred embodiment.

[0053] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H3" having a VL with an amino acid sequence of SEQ ID NO: 8 and a VH with an amino acid sequence of SEQ ID NO: 11 is a third more preferred embodiment.

[0054] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H4" having a VL with the amino acid sequence of SEQ ID NO: 8 and a VH with the amino acid sequence of SEQ ID NO: 12 is a fourth more preferred embodiment.

[0055] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H5" having a VL with the amino acid sequence of SEQ ID NO:9 and a VH with the amino acid sequence of SEQ ID NO:11 is a fifth more preferred embodiment.

[0056] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H6" having a VL with the amino acid sequence of SEQ ID NO:9 and a VH with the amino acid sequence of SEQ ID NO:12 is the sixth more preferred embodiment.

[0057] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H7" having a VL with an amino acid sequence of SEQ ID NO: 10 and a VH with an amino acid sequence of SEQ ID NO: 11 is the seventh more preferred embodiment.

[0058] In the preferred embodiment of the first aspect of the present invention, characterized in that the CAR "H8" having a VL with the amino acid sequence of SEQ ID NO: 10 and a VH with the amino acid sequence of SEQ ID NO: 12 is an eighth more preferred embodiment.

[0059] The most preferred CARs among the above more preferred embodiments of the first aspect of the invention are those of the first and second more preferred embodiments ("H1" and "H2").

[0060] The CAR of this first aspect of the invention is not limited to the specific peptide sequences provided. On the contrary, the invention also includes variants. With reference to the present disclosure and conventionally available techniques and references, a skilled person will be able to prepare, test and utilize functional variants of the CAR disclosed herein, while recognizing that having a binding affinity for IGLV3-21 R110 -These variants of the BCR and thereby the ability to kill B cells are within the scope of the present invention.

[0061] Variants can include, for example, CARs having at least one altered complementarity determining region (CDR) (hypervariable) and / or framework (FR) (variable) region / position relative to a peptide sequence disclosed herein.

[0062] As an example, a skilled artisan can use the sequences of the CARs provided herein (e.g., of Table 3) to design variants that fall within the scope of the present invention.

[0063] Furthermore, by using a CAR of the first aspect of the invention as a starting point for optimization, variants are obtained by diversifying one or more amino acid residues in the CAR, preferably amino acid residues in one or more CDRs, and by screening the set of obtained CAR variants.

[0064] Polypeptide variants of the CAR binding domain can be prepared that retain the overall molecular structure of the CAR described herein. Considering the properties of the individual amino acids, the skilled person will recognize some reasonable substitutions. For example, amino acid substitutions, "conservative substitutions", can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues involved.

[0065] For example, (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; (c) positively charged (basic) amino acids include arginine, lysine and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions can generally occur within groups (a)-(d). In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine and proline usually appear in that order. Some preferred substitutions can be made between the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can easily construct DNA encoding such conservative amino acid variants.

[0066] The CAR according to the first aspect of the present invention may also include a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0067] The CAR according to the first aspect of the present invention may also include a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

[0068] Humanized anti-IGLV3-21 of the CAR according to the first aspect of the present invention R110 The binding domain is preferably a scFv. The order of VL and VH in the scFv can be VL-VH or VH-VL from N-terminus to C-terminus, wherein the preferred order is VL-VH. The VL of the scFv is preferably attached to VH via a suitable amino acid linker. The preferred scFv of the CAR according to the first aspect of the present invention has an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0069] The CAR according to the first aspect of the present invention may also include a transmembrane domain derived from a type 1 transmembrane protein (such as CD28, CD3ζ, CD4, CD8α or OX40). Preferably, the transmembrane domain is human. Specifically, the transmembrane domain may be derived from human CD28.

[0070] The CAR according to the first aspect of the present invention may also include a spacer region. Generally, a spacer is necessary to isolate the antigen binding domain from the membrane to provide a path and certain flexibility for contacting the antigen. The spacer may be derived from IgG1 or IgG4 or from extracellular CD28, CD4 or CD8. Specifically, the spacer may be the CH1-CH2-CH3 constant region of IgG1 or IgG4. More specifically, a more compact spacer may be sufficient, such as an IgG1 or IgG4 hinge. Preferably, the spacer may be human.

[0071] According to a first aspect of the present invention, the cytoplasmic domain may include a signaling domain, consist essentially of a signaling domain, or consist of a signaling domain. The signaling domain may include one or more tyrosine-based activation motifs, preferably an immunoreceptor tyrosine activation motif (ITAM). Once the conserved tyrosine residues within this motif are phosphorylated, the ITAM serves as a specific adapter for downstream signaling proteins. The signaling domain may be derived from, for example, but not limited to, CD3ζ, FcεR1, DAP10 or DAP12. The FcεR1 signaling domain includes one ITAM, and the CD3ζ signaling domain includes three ITAMs and binds to a T cell receptor to generate a signal. The signaling domain may be or include a T cell signaling domain. Preferably, the signaling domain may be of human origin. Specifically, the signaling domain may be derived from human CD3ζ.

[0072] The cytoplasmic domain may include one or more additional costimulatory domains. Suitable costimulatory domains may include domains derived from CD2, CD27, CD28, 4-1BB (CD137), CD244, ICOS or OX40, which may be used alone or in any combination thereof. Therefore, the cytoplasmic domain may include only a signaling domain, or a combination of a signaling domain and one or more costimulatory domains. Specifically, the cytoplasmic domain may include a combination of a CD3 ζ signaling domain and costimulatory domains CD28 and 4-1BB.

[0073] The humanized CAR according to the first aspect of the present invention specifically binds to IGLV3-21 R110(SEQ ID NO: 41 and Figure 14). R110 Positive and IGLV3-21 R110 Negative cell lines and primary CLL cells demonstrated that, unlike anti-CD19 CAR, humanized anti-IGLV3-21 R110 CAR effectively distinguishes wild-type IGLV3-21 G110 Variant and malignant IGLV3-21 R110 Variants (Example 3, Figures 3, 4, 6, and 9).

[0074] Furthermore, as described herein, it has been shown that humanized anti-IGLV3-21 R110 CAR T cells selectively kill IGLV3-21 R110 Positive cell lines and primary CLL cells, but not polyclonal non-malignant B cells, however, these cells are also affected by the cell killing of anti-CD19 CAR T. Therefore, the beneficial aspect of the present invention is that humanized anti-IGLV3-21 R110 CAR specifically and selectively binds IGLV3-21 R110 positive B cells, and thereby being able to selectively kill malignant cells while retaining healthy human B cells (Example 3; Figure 7 ).

[0075] It was observed that the use of anti-IGLV3-21 based T cells derived from healthy donors R110 CAR T cells have better killing (for example, Example 4 and Fig.11 ), and even CAR T cells derived from CLL patients showed effective killing capacity, although it is well known that T cells in CLL are functionally impaired (Examples 4 and Fig.11 ).

[0076] In addition, the killing of CLL B cells is accompanied by an increase in the levels of immune effector cytokines (Examples 5 and 6, Figures 5, 8, Fig.12 ).

[0077] In summary, it has been demonstrated that humanized anti-IGLV3-21 R110 CAR for precise targeting of IGLV3-21 using CAR T cells R110 Excellent applicability of positive B cells. This precision and their low immunogenicity make the humanized CARs of the present invention a potential tool in the field of treatment of CLL patients, who are characterized by a compromised immune system due to the underlying disease.

[0078] In addition to the above IGLV3-21 R110In addition to the definitions provided, in generally preferred embodiments of this first aspect of the invention, the IGLV3-21 R110 is further characterized in that it has greater than 80% sequence identity with the sequence represented by SEQ ID NO 41, wherein there is a lysine at position 16 and aspartic acid at positions 50 and 52. In a generally more preferred embodiment of this first aspect of the invention, there is a tyrosine or a phenylalanine at position 49 and a serine or a threonine at position 51. In a generally further more preferred embodiment of this first aspect of the invention, there is a tyrosine at position 49 and a serine at position 51.

[0079] Second Aspect of the Invention - Polynucleotides

[0080] The present invention also relates to a polynucleotide encoding the CAR of the first aspect of the present invention. The polynucleotide according to the present invention may comprise DNA or RNA.

[0081] DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants in the present invention can be described with reference to their physical properties in hybridization. The technician will recognize that DNA can be used to identify its complement, and because DNA is double-stranded, nucleic acid hybridization techniques can be used to identify its equivalent or homologue. It will also be recognized that hybridization can occur when complementarity is less than 100%. However, assuming that the conditions are selected appropriately, hybridization techniques can be used to distinguish DNA sequences according to the structural correlation of DNA sequences with specific probes. For guidance on such conditions, see Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & S1ruhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0082] The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences will hybridize to each other. As used herein, the term "stringency" refers to the degree to which the conditions are unfavorable for hybridization. Stringent conditions are very unfavorable for hybridization, and only molecules with the most related structures will hybridize to each other under such conditions. In contrast, non-stringent conditions are favorable for the hybridization of molecules with a lower degree of structural correlation displayed. Therefore, hybridization stringency is directly related to the structural relationship of the two nucleic acid sequences. The following relationship can be used to correlate hybridization and correlation (where Tm is the melting temperature of a nucleic acid duplex): aT m =69.3+0.41(G+C)% b. For every 1% increase in the number of mismatched base pairs, the T m Reduce by 1℃ c.(T m )μ2-(T m )μ1=18.5log 10 μ2 / μ1 where μ1 and μ2 are the ionic strengths of the two solutions.

[0083] Hybridization stringency is a function of many factors, including total nucleotide concentration, ionic strength, temperature, probe size, and the presence of agents that disrupt hydrogen bonding. Factors that promote hybridization include high nucleotide concentration, high ionic strength, low temperature, longer probe size, and the absence of agents that disrupt hydrogen bonding. Hybridization is usually carried out in two stages: a "binding" stage and a "cleaning" stage.

[0084] Yet another class of polynucleotide variants within the scope of the present invention can be described with reference to the products they encode. It will be appreciated that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequences encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the product will be expressed. These functionally equivalent polynucleotides are characterized in that, due to the degeneracy of the genetic code, they encode the same peptide sequences found in SEQ ID NOs: 1 to 20.

[0085] It has been recognized that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they can be constructed into fully synthetic DNA. Methods for effectively synthesizing oligonucleotides within the range of 20 to about 150 nucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides can be synthesized and assembled in the manner first reported in Khorana et al., J.Mol.Biol.72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. Preferably, the synthetic DNA is designed to have convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into a suitable vector.

[0086] As shown, a method for generating variants starts with one of the DNAs disclosed herein and then performs site-directed mutagenesis. See Ausubel et al., chapter 8, Supplement 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA phage vector. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide changes. The complementary strand is synthesized and the double-stranded phage is introduced into the host. Some of the offspring produced will contain the desired mutants, which can be confirmed using DNA sequencing. In addition, various methods can be used to increase the possibility that the offspring phage will be the desired mutant. These methods are well known to those skilled in the art, and commercially available kits are used to generate such mutants.

[0087] As described in the first aspect of the present invention, the second aspect of the present invention also has corresponding preferred embodiments.

[0088] In a first preferred embodiment of this second aspect of the invention, the present invention relates to a DNA molecule encoding a CAR, which comprises a sequence as shown in SEQ ID NO: 21 (indicating L-CDR1), SEQ ID NO: 22 (indicating L-CDR2), SEQ ID NO: 23 (indicating L-CDR3), SEQ ID NO: 24 (indicating H-CDR1), SEQ ID NO: 25 (indicating H-CDR2) and SEQ ID NO: 26 (indicating H-CDR3).

[0089] In a second preferred embodiment of this second aspect of the invention, the present invention relates to a DNA molecule encoding a CAR, which comprises a VL sequence as shown in any one of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0090] In a third preferred embodiment of the second aspect of the invention, the present invention relates to a DNA molecule encoding a CAR, which comprises a VH sequence as shown in any one of SEQ ID NO:31 and SEQ ID NO:32.

[0091] In a fourth preferred embodiment of this second aspect of the invention, the present invention relates to a DNA molecule encoding a CAR, which comprises a scFv sequence as shown in any one of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

[0092] The third aspect - carrier

[0093] The third aspect of the present invention relates to a vector comprising the polynucleotide according to the second aspect of the present invention.

[0094] Such vectors can be used to introduce polynucleotides into host cells so that they express the CAR according to the first aspect of the invention.

[0095] The vector may be a plasmid or a viral vector, preferably a lentiviral or retroviral vector.

[0096] Methods for packaging polynucleotides into plasmids or viral vectors are known in the art.

[0097] The vector can be used to transduce or transfect cells.

[0098] In addition to the gene of interest, the vector may also encode a reporter gene that allows identification, detection and / or selection of the expressed gene of interest in a given host cell using molecular biological and immunological methods known in the art.

[0099] Such a reporter gene can be a truncated epidermal growth factor receptor (EGFRt) for antibody staining and sorting of cells expressing CAR in flow cytometry. In order to co-express truncated EGFR, the vector can also encode a viral self-cleaving peptide, such as T2A.

[0100] Aspect 4 - Cells

[0101] A fourth aspect of the present invention relates to cells expressing CAR according to the first aspect of the present invention. The cell may be an immune effector cell, such as a T cell, such as an initial T cell, a central memory T cell, an effector memory T cell, a natural killer (NK) cell, or a cytokine-induced killer cell. Preferably, the cell may be a human T cell.

[0102] The fourth aspect also relates to an in vitro method for preparing cells expressing CAR, which comprises introducing the polynucleotide according to the second aspect of the present invention or the vector according to the third aspect of the present invention into immune cells according to the fourth aspect of the present invention under suitable conditions.

[0103] Fifth aspect - pharmaceutical composition

[0104] The fifth aspect of the present invention relates to a pharmaceutical composition, which includes the CAR of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention, the vector of the third aspect of the present invention, or the cell of the fourth aspect of the present invention and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers may be, for example, vehicles, excipients, diluents, and adjuvants. Acceptable carriers are nontoxic to the recipient and include, but are not limited to, stabilizers, buffers (such as phosphates, citrates, or other organic acids), saline, buffered saline, low molecular weight polypeptides, proteins (such as serum albumin), any sterile, biocompatible drug carriers, glucose, and water. Optionally, the pharmaceutical composition may include other pharmaceutically active polypeptides and / or compounds.

[0105] The pharmaceutical composition according to the fifth aspect of the present invention may be manufactured in a manner known in the art, for example, by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes.

[0106] After the pharmaceutical compositions have been prepared, they can be placed in suitable containers and labeled for use in treating the indicated condition, wherein the pharmaceutical compositions include the CAR of the first aspect of the invention, the polynucleotide of the second aspect of the invention, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention formulated in a suitable carrier.

[0107] Such administration is usually accomplished by parenteral administration. Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

[0108] Preferred routes of administration are intravenous and intraarterial (directly to the tumor).

[0109] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical compositions of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiologically buffered saline. Water-soluble injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Alternatively, suspensions of the active compounds may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include: fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.

[0110] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0111] Aspect 6 - Medical Use

[0112] The sixth aspect of the present invention relates to the use of the CAR of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention, or the pharmaceutical composition of the fifth aspect of the present invention in treating a disease. Specifically, the sixth aspect of the present invention relates to the use of the cell of the fourth aspect of the present invention in treating a disease.

[0113] Similarly, the sixth aspect of the present invention relates to a method for treating a disease, comprising administering to a subject in need thereof the CAR of the first aspect of the present invention, the polynucleotide of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention, or the pharmaceutical composition of the fifth aspect of the present invention. Specifically, the method according to the sixth aspect of the present invention may include administering to a subject the cell of the fourth aspect of the present invention.

[0114] The method according to the sixth aspect of the present invention may comprise the following steps: i) providing immune cell populations, ii) introducing the polynucleotide according to the second aspect of the present invention or the vector according to the third aspect of the present invention into the immune cell, iii) culturing the immune cells under conditions that allow expression of the CAR, and administering the cells from (iii) to a subject. Suitably, the cells may be autologous or allogeneic.

[0115] According to this sixth aspect of the invention, the disease may be chronic lymphocytic leukemia (CLL). Specifically, the disease to be treated is clinically manifested CLL caused by abnormal proliferation of B cells with autonomous active BCRs. More specifically, the disease is clinically manifested CLL characterized by having cells carrying IGLV3-21 R110 Abnormal proliferation of B cells with autonomously activated BCR.

[0116] As discussed in relation to the first aspect of the present invention, it has been shown that humanized anti-IGLV3-21 R110 CAR specifically and selectively mediates the expression of IGLV3-21 in CAR T cells R110 The toxicity of the humanized anti-IGLV3-21 according to the present invention is to kill malignant B cells with positive BCR. R110 The applicability of CAR for precision therapy of CLL with CAR T cells.

[0117] According to the sixth aspect of the present invention, the CAR according to the first aspect of the present invention, the polypeptide according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the cell according to the fourth aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention can also be administered to the subject in combination with another therapeutic agent. Therefore, the CAR, the polypeptide, the vector, the cell, or the pharmaceutical composition can be administered as a single agent, or in combination with one or more additional therapeutic agents, wherein the combination does not cause unacceptable adverse reactions. The combination therapy includes administering a separate pharmaceutical preparation, the separate pharmaceutical preparation comprising the CAR, the polypeptide, the vector, the cell, or the pharmaceutical composition, and one or more additional therapeutic agents; and administering the CAR, polypeptide, vector, cell, or pharmaceutical composition of the present invention, and each additional therapeutic agent in its respective separate pharmaceutical preparation.

[0118] When using a separate formulation, the treatment according to the sixth aspect of the present invention and one or more other therapeutic agents can be carried out substantially simultaneously (e.g., simultaneously) or at separate staggered times (e.g., sequentially). Specifically, the treatment according to the present invention can be carried out in combination with other antitumor agents (e.g., alkylating agents, antimetabolites, antitumor agents of plant origin, hormone therapeutic agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and / or biological response modifiers, anti-angiogenic compounds and other antitumor drugs) fixedly or individually.

[0119] A preferred additional therapeutic agent for co-administration according to the sixth aspect of the invention is a Bruton's tyrosine kinase (BTK) inhibitor.Preferably, the BTK inhibitor may be co-administered with the cells according to the fourth aspect of the invention, wherein the cells may be autologous.

[0120] Certain Bruton's tyrosine kinase (BTK) inhibitors are commercially available. It is known to induce apoptosis in B cell lymphoma and CLL cells (Hermann SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011; 117: 6287-6296). Other BTK inhibitors include, for example, ACP-196 from Acerta Pharma BV and BGB-3111 from BeiGene, Co., Ltd. Preferably, the additional therapeutic agent is ibrutinib. As shown in the examples herein, the impaired functionality of CAR T cells derived from CLL patients can be fully restored by subtherapeutic doses of ibrutinib (Example 4, Fig.11 ).

[0121] Further aspects of the invention - kits

[0122] The present invention also relates to pharmaceutical packaging and kits comprising one or more containers containing one or more ingredients of the pharmaceutical composition according to the fifth aspect of the present invention. Associated with such containers may be a notice in a form prescribed by a governmental agency governing the manufacture, use or sale of pharmaceutical products or biological products, reflecting the agency's approval of the manufacture, use or sale of the product for human administration.

[0123] The preferred embodiments of the present invention are: A. Humanized chimeric antigen receptor (CAR), comprising humanized IGLV3-21 R110 Binding domain, transmembrane domain and cytoplasmic domain, wherein the humanized IGLV3-21 R110The binding domain includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) includes a light chain complementary determining region 1 (L-CDR1) having an amino acid sequence of SEQ ID NO: 1, a light chain complementary determining region 2 (L-CDR2) having an amino acid sequence of SEQ ID NO: 2, and a light chain complementary determining region 3 (L-CDR3) having an amino acid sequence of SEQ ID NO: 3, and the heavy chain variable region (VH) includes a heavy chain complementary determining region 1 (H-CDR1) having an amino acid sequence of SEQ ID NO: 4, a heavy chain complementary determining region 2 (H-CDR2) having an amino acid sequence of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (H-CDR3) having an amino acid sequence of SEQ ID NO: 6. B. The CAR according to embodiment A, characterized in that the VL having an amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 and Any combination of VH having an amino acid sequence selected from SEQ ID NO:11 and SEQ ID NO:12. C. A CAR according to embodiment B, characterized in that it has a VL having an amino acid sequence of SEQ ID NO: 7 and a VH having an amino acid sequence of SEQ ID NO: 11, or a VL having an amino acid sequence of SEQ ID NO: 7 and a VH having an amino acid sequence of SEQ ID NO: 12. D. A CAR according to any of the preceding embodiments, characterized in that the VL has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity with the amino acid sequence of the VL according to embodiment C, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity with the amino acid sequence of the VH according to embodiment C. E. A CAR according to any one of the preceding embodiments, wherein the humanized anti-IGLV3-21 R110 The binding domain is a scFv. F. The CAR according to embodiment E, wherein the scFv has an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. G. A polynucleotide encoding a CAR according to any one of the preceding embodiments. H. A vector comprising a polynucleotide according to embodiment G. I. A cell expressing a CAR according to any one of embodiments A to F. J. A cell according to embodiment I, wherein the cell is a T cell. K. A pharmaceutical composition comprising a CAR according to any one of embodiments A to F, a polynucleotide according to embodiment G, a vector according to embodiment H or a cell according to embodiment I or J, and a pharmaceutically acceptable carrier. L. Use of the CAR according to any one of embodiments A to F, the polynucleotide according to embodiment G, the vector according to embodiment H, the cell according to embodiment I or J, or the pharmaceutical composition according to embodiment K in treating a disease. M. A method for treating a disease, comprising the step of administering to a subject in need thereof a CAR according to any one of embodiments A to F, a polynucleotide according to embodiment G, a vector according to embodiment H, a cell according to embodiment I or J, or a pharmaceutical composition according to embodiment K. N. A method according to embodiment M, comprising the following steps: iv) providing immune cell populations, v) introducing the polynucleotide according to embodiment G or the vector according to embodiment H into said immune cell, vi) culturing the immune cells under conditions that allow expression of the CAR, and vii) administering the cells from (iii) to a subject. O. Use of the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to embodiment L or the method according to embodiment M or N, wherein the disease is chronic lymphocytic leukemia (CLL). P. The use of the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to embodiment L or O, or the method according to any one of embodiments M to O, wherein the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition is to be administered in combination with an additional therapeutic agent. Q. Use of the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to embodiment P, wherein the additional therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor. R. A kit comprising a pharmaceutical composition according to embodiment K. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 Humanized anti-IGLV3-21 is shown R110 Schematic diagram of CAR.

[0125] Figure 2A and Figure 2B Shown is a method for expressing humanized anti-IGLV3-21 R110 CAR lentiviral vectors pJ2459_h1_R110-1BB and pJ2487_h1_R110-1BB.

[0126] FIG. 3 shows representative images of a cytotoxicity assay according to Example 3 after 24 hours of co-incubation. Figure 3A and Figure 3B Figure 3 shows the expression of anti-IGLV3-21 from healthy donors expressing humanized scFv H1 (3A: CARHDh1) or scFv H2 (3B: CARHDh2). R110 Determination of CAR T cells and those derived from CLL without IGHV3-21 R110 - Patients with BCR-characterized (i.e., non-IGLV3-21 R110 Determination of OCI-LY1_wt cells (OCI wt) with CLL). Figure 3D and Figure 3E The expression of IGLV3-21 was expressed using R110 The same assay was performed on the cell line OCI-LY1_R110 (OCIR110). Figure 3A and Figure 3D as well as Figure 3B and Figure 3E Comparison of the results shows that anti-IGLV3-21 from a healthy donor with humanized scFv H1 and scFv H2, as indicated by the large bright clusters, R110 CAR T cells show expression of IGLV3-21 R110 Selective killing of the cell line OCI-LY1_R110. Figure 3C and Figure 3F A control with non-transduced CAR T cells from a healthy donor is depicted.

[0127] FIG. 4 shows a graph of a killing assay of CAR T cells performed according to Example 3. Figure 4A Depicts the results of co-incubation of the indicated OCI-LY1_wt cells (OCI wt) with CAR T cells derived from different healthy donors; Figure 4B The same set of assays for OCI-LY1_R110 cells is shown in FIG. Figure 4A It can be seen that OCI-LY1_wt cells were killed by anti-CD19CAR T cells (CARHDCD19), but not by humanized anti-IGLV3-21 R110The effect of CAR (CARHDh2) on CAR T cells. Figure 4A and Figure 4B It is easy to see that, unlike anti-CD19 CAR T cells, anti-IGLV3-21 R110 CAR T cells selectively kill IGLV3-21 R110 Positive cell line OCI-LY1_R110.

[0128] Figure 5 shows a graphical representation of cytokine quantification in the 24 hour co-culture supernatants of the killing assay depicted in Figure 4. Figure 5A and Figure 5B As can be seen, in the supernatant showing the cell killing ability of CAR T cells, the IFN-γ secretion level and the IL-6 secretion level were increased.

[0129] FIG. 6 shows a graphical representation of the killing calculated from the killing assay of CAR T cells after 24 hours of co-incubation according to Example 3. Fig. 6A Depicted are the expression of anti-IGLV3-21 derived from healthy donors as compared to untransduced cells (CARHDUTD). R110 CAR T cells (CARHDh2), anti-CD19 CAR T cells (CARHDCD19), and anti-TSHR control CAR T cells (CARHDTSHR) from IGLV3-21 R110 Killing activity of primary CLL cells from negative CLL cases (CLL426 and CLL427). Figure 6B Describes the Fig. 6A The same as shown in R110 Analysis of killing of primary CLL cells from positive CLL cases (CLL438, CLL442). Fig. 6A and Figure 6B As shown in the comparison, CD19-directed CAR T cells kill from IGLV3-21 R110 Positive and IGLV3-21 R110 Negative CLL cases both primary CLL cells, and anti-IGLV3-21 R110 CAR T cells selectively recognize and kill IGLV3-21 R110 Positive CLL cells.

[0130] Figure 7A graph of a CAR T cell killing assay is shown, which quantifies polyclonal B cell killing mediated by CAR T cells derived from healthy donors compared to untransduced cells (CARHDUTD). The assay was performed according to Example 3. As can be seen from the figure, polyclonal healthy B cells were killed by anti-CD19 CAR T cells (CARHDCD19), but not by anti-IGLV3-21 R110 Effects of CAR T cells, demonstrating epitope-specific targeting with preservation of normal B cells.

[0131] FIG8 shows FIG6 and Figure 7 Graphical representation of cytokine quantification in 24-hour co-culture supernatants from the killing assay depicted in FIG. Fig. 8A and Figure 8B As can be seen, in the supernatant showing cell killing by CAR T cells, both the IFN-γ secretion level and the IL-6 secretion level were increased.

[0132] FIG. 9 shows a graphical representation of the killing calculated from the cell killing assay of CAR T after 24 hours of co-incubation according to Example 3. Fig.9A Figure 3 shows the expression of anti-IGLV3-21 in CLL patient-derived cells compared with untransduced cells (CARCLLUTD). R110 Quantification of OCI-Ly1_wt (OCI wt) killing mediated by CAR T cells expressing CAR (CARCLLh2) or anti-CD19 CAR (CARCLLCD19), and Fig. 9B Quantification of OCI-Ly1_R110 (OCIR110) killing is depicted. These assays were performed with two CLL patients as T cell donors, one with active CLL (CLL433) and the other with CLL in remission (CLL453). Fig.9A and Fig. 9B It can be clearly seen from the comparison that the anti-IGLV3-21 R110 CAR T cells showed selective killing of Oci-Ly1-R110 (OCIR110), while anti-CD19 CAR T cells from the same patient killed regardless of the neo-epitope.

[0133] Fig.10Determination of subtherapeutic doses of ibrutinib is shown. The figure depicts that after serial dilutions of ibrutinib (11 steps, ranging from 0.01 to 20 μM) were applied to OCI-Ly1wt or OCI-Ly1_R110 cells for 24 hours, ibrutinib was titrated to determine toxicity to the OCI-Ly1 cell model. The percentage of cell growth is plotted as the total number of ibrutinib-treated cells relative to the total number of untreated cells. This analysis revealed that 0.08 μM ibrutinib as the highest dose did not result in a decrease in target cell viability. Therefore, this dose was selected for further testing of the efficacy of CAR T.

[0134] Fig.11 Figure 4 shows a calculated killing graph from a cell killing assay of CAR T cells according to Example 4, showing the effect of a subtherapeutic dose of the BTK inhibitor ibrutinib; all cells and ibrutinib were added as indicated in the graph. The graph depicts the effect of a subtherapeutic dose of the BTK inhibitor ibrutinib at ...; all cells and ibrutinib were added as indicated in the graph. R110 CAR T cells (CARHDh2) and anti-IGLV3-21 from the blood of CLL patients R110 CAR T cells (CLL425, CARCLLh2) expressing IGLV3-21 R110 Therefore, using T cells derived from healthy donors based on anti-IGLV3-21 R110 CAR T cells derived from CLL patients also showed effective killing ability. In addition, polyclonal B cells (B HD ) were not affected by these CAR T cells, indicating that both CAR T cells from healthy donors and CAR T cells from CLL patients were able to selectively kill IGLV3-21 R110 positive cells, while sparing healthy B cells. In addition, the killing capacity of CAR T cells derived from healthy donors was not altered in the presence of subtherapeutic doses of ibrutinib throughout the 16-hour monitoring period, as shown by a comparison of cytotoxicity with and without ibrutinib. In the absence of ibrutinib, the killing capacity of CAR T cells derived from CLL patients decreased over time, while the presence of ibrutinib increased the killing capacity of cells derived from CLL patients to the level of healthy donor CAR-T cells throughout the monitoring period.

[0135] Fig.12 Shows Fig.11 Graphical representation of the cytokine IFN-γ secreted by CAR T cells in the supernatant of co-culture for 16 hours in the killing assay described in . Fig.12It can be seen that elevated IFN g levels were measured in the corresponding supernatants showing cell killing by CAR T cells.

[0136] Fig.13 Depicted IGLV3-21 R110 Schematic diagram of BCR-BCR homotypic interaction of light chain (as described by Minici et al.). Two adjacent BCRs are depicted as having an antigen binding subunit including a heavy chain (HC) and a light chain (LC), a transmembrane domain (TM), and a signal transduction subunit (SU) composed of a heterodimer of Igα and Igβ proteins (CD79a / CD79b) connected by disulfide bonds. The mutant arginine (R) at position 110 of a BCR interacts with the germline-encoded aspartic acid (D) at position 50 of the adjacent BCR. Further interactions between the two BCRs are mediated by the germline-encoded amino acid residues lysine (K) at position 16 and aspartic acid (D) at position 52.

[0137] Figure 14 shows IGLV3-21 R110 Schematic diagram of . Fig.14A IGLV3-21: Exemplary single letter codons R110 (SEQ ID NO:41). Amino acid residues involved in BCR-BCR homotypic interactions according to Minici et al. are marked in bold. Fig. 14B : Row 1: IGLV3-21 R110 Row 2: Amino acid residues involved in BCR-BCR homotypic interactions according to Minici et al. Row 3: IGLV3-21 R110 The YDSD motif in . Amino acids are described by 3-letter codes.

[0138] Fig.15 The sequences according to SEQ ID No: 1 to 55 are shown. DETAILED DESCRIPTION

[0139] The present invention is further described by the following examples. Examples are provided only to illustrate the present invention by reference to specific embodiments. Although these examples illustrate certain specific aspects of the present invention, they do not reflect the limitations of the disclosed invention and do not limit its scope.

[0140] All examples are implemented using standard techniques, which are well known and routine to those skilled in the art, unless otherwise described in detail. The conventional molecular biology techniques of the following examples can be performed as described in standard laboratory manuals (such as Sambrook et al., 1989, supra).

[0141] The preferred embodiments of the present invention are:

[0142] Example

[0143] Example 1

[0144] Derived from humanized scFv and humanized anti-IGLV3-21 R110 CAR mouse anti-IGLV3-21 R110 Antibody design

[0145] 1. Antibodies

[0146] 1.1. Mouse monoclonal antibody mAb01-01

[0147] By combining IGLV3-21 with a soluble form R110 Mouse monoclonal antibody mAb01-01 was developed by immunizing mice with the BCR of WT BCR (see SEQ ID NOs: 42 and 43) and selecting appropriate antibodies using a cell-based system in which the intact, functional BCR is presented membrane-bound.

[0148] First, IgG 1 Therefore, DNA fragments encoding IGHV3-21 were synthesized by a contract manufacturer using standard procedures, as were DNA for an exemplary variable heavy chain (VH) and DNA for a complete light chain (LC) to cover the entirety of IGLV3-21. R110 They were then compared with mouse IgG by polymerase chain reaction (PCR). 1 The constant segments were fused and cloned into a cytomegalovirus (CMV) vector. In the Journal of Clinical Pharmacology, 2.Auflage, SpringerVerlag 2019, a human cell expression system based on HEK293T cells was used to express such IgG 1 (SEQ ID NO:42 represents VH, and SEQ ID NO:43 represents LC). Transfection was performed using a polyethyleneimine (PEI)-based protocol. After several passages, the supernatants were pooled and the culture medium contained in the combined cell supernatants was purified using a protein G column. Soluble IgG was determined by Western blotting. 1 purity and quality.

[0149] Thereafter, mice were immunized with a recombinantly produced soluble form of the BCR (see SEQ ID NOs: 42 and 43).

[0150] Immune cells with the desired specificity can then be obtained from these mice and converted into hybridoma cells by cell fusion. Monoclonal antibodies are produced in mice using standard procedures, followed by the generation of hybridoma cells.

[0151] Different from the usual screening of positive clones by enzyme-linked immunosorbent assay (ELISA). Because the target structure is a membrane-bound receptor, it is crucial to verify the combination of potential antibodies in the cell system, that is, to retain the cell physiological state of this cell type to a great extent at the same time. First, the binding events of the supernatants collected by multiple groups are checked using fluorescence activated cell sorting (FACS) analysis. For this purpose, different BCR variants are expressed on the surface of the cell line of triple knockout (triple knockout, TKO), and the cell line itself cannot express BCR.

[0152] The starting point for the generation of TKO cells is formed by transgenic mice that knock out the genes Lambda5, RAG1 or RAG2 and SLP65, respectively (Dühren von Minden et al., 2012, Nature 489, p.309-313). The knockout combination of RAG2 or RAG1 and Lambda5 leads to an obstruction of the transformation from the pro-B cell stage to the pre-B cell stage, which is usually characterized by the beginning of the rearrangement of the VDJ fragment of the heavy chain (HC). Therefore, they are pro- / pre-B cells. The activity of BCR can be measured by reconstruction with inducible SLP65. The generation of such mice is known to experts and belongs to the prior art. In order to obtain the cells, after the mice are killed, the femoral bone marrow is extracted from the mice. The cells obtained in this way are then cultured under conditions that promote the survival of pro- / pre-B cells (37°C, 7.5% CO 2 , Iscoves medium, 10% FCS, P / S, murine IL7). After several passages, FACS sorting is performed for control purposes, and progenitor / pre-B cells are sorted and then returned to culture. Markers for this purpose are known to the expert.

[0153] In order to reconstruct with "BCR of interest", the corresponding coding sequence of VH is fused with human IgM constant fragment by polymerase chain reaction (PCR), and heavy chain (HC) and light chain (LC) are cloned into corresponding expression vectors each having CMV promoter. By lipofection, they are introduced into packaging cell line (Phoenix cell line). After incubation for 36 hours, the viral supernatant is taken out and used for centrifugal infection (spinfection) of TKO cells. BCR expression is measured using anti-IgM and anti-LC antibodies on FACS. For this purpose, some cells are taken out and stained with 5 μl of antibody per 100 μl total volume of PBS. Both the work of extracting supernatant and the centrifugal infection of TKO are well-known procedures and are known to experts. The knockout of RAG2 or RAG1 and Lambda5 ensures that only "BCR of interest" is expressed on the surface.

[0154] In this way, two different BCR-expressing TKO cell lines were generated, one of which expressed membrane-bound IGHV3-21 / IGLV3-21 R110 To generate a second BCR-expressing TKO cell line, the gene encoding IGLV3-21 was transfected into the cell line using the well-known site-directed mutagenesis technique (see, e.g., Sambrook et al., 1989 supra). R110 The arginine codon at position 110 of the DNA was restored to the germline sequence. The resulting TKO cells expressed a BCR containing IGLV3-21, in which the 110th amino acid was glycine (IGLV3-21 G110 ). To generate a third control TKO cell line that does not express the BCR on its surface, an empty expression vector was used for centrifugal infection. By reconstituting the cells with inducible SLP65, the function of the expressed BCR can be characterized and thus the expression of IGHV3-21 / IGLV3-21 on the surface can be verified before selection. R110 The method chosen here is to measure the Ca flux after SLP65 induction using FACS analysis and to use Ca 2+ These methods are known to experts (see M. Dühren-von Minden et al.; Nature 2012). Using these cells as “targets”, FACS has now been used to identify cells that specifically bind to the IGLV3-21 gene. R110 The first step is to identify supernatants to which the antibody shows binding. In the first round of selection, supernatants from several clones are pooled and examined for their binding profiles. If a clone shows binding to IGHV3-21 / IGLV3-21 R110-BCR specific binding, then show its positive binding profile. Groups showing such profiles were isolated and the binding profiles of individual clones were characterized again in a second round of selection. The binding of the monoclonal antibodies was verified using a FACS binding assay using a fluorescently labeled anti-mouse IgG antibody. This method allowed the isolation of the monoclonal antibody mAb01-01, which showed specific binding to human IGLV3-21 R110 -High specificity and cross-reactivity of the BCR, but wild-type IGLV3-21 carries a germline-encoded glycine at amino acid position 110 G110 The variant did not have any detectable binding.

[0155] Antibody mAb01-01 was sequenced. The CDRs were identified by amino acid annotation based on the IMGT numbering system. SEQ ID NOs: 1, 2, and 3 represent the sequences corresponding to the CDRs of the light chain (L-CDR1, L-CDR2, and L-CDR3), while SEQ ID NOs: 4, 5, and 6 represent the sequences corresponding to the CDRs of the heavy chain (H-CDR1, H-CDR2, and H-CDR3). The amino acid sequences of the murine variable light and variable heavy chains were identified as SEQ ID NO: 44 for VL, and SEQ ID NO: 45 for VH.

[0156] 1.2. Humanized anti-IGLV3-21 R110 Antibody

[0157] 1.2.1 Humanization of mAB01-01

[0158] Humanization of VL and VH was performed by in silico transplantation of mouse CDRs into mature human variable light and heavy chain regions using standard CDR transplantation techniques from Fusion Antibodies Plc, Belfast, N.Ireland. Many human framework sequences have been identified for use as "acceptor" frameworks for mAB01-01 CDR sequences. These acceptor sequences are all derived from mature human IgG from humans. Therefore, it is expected that the humanized sequences are non-immunogenic and retain the typical structure of the CDR loops.

[0159] The V region closest to the human germline gene of the mouse VH domain is Homo sapiens IGHV4-59. The V region closest to the human germline gene of the mouse VL domain is Homo sapiens IGKV1-9. New variants are designed based on variants that show binding in previous variants. Germlines similar to the parent antibody but with more differences in the framework are also selected, CDRs are transplanted and back mutations are introduced to maintain binding. Using the CDRx platform, key residues important for VL / VH interface and typical loop structure are retained as much as possible in the humanized variants (Fusion Antibodies Plc, Belfast, N.Ireland).

[0160] Humanized variants are examined to determine whether they have been humanized according to the WHO definition of humanized antibodies (see also World Health Organization (WHO) International Nonproprietary Names (INN) for biological and biotechnological substances (a review) 2014): the variable region of the humanized chain has a V region amino acid sequence (usually derived from a V region gene segment after somatic hypermutation) that, when analyzed as a whole, is closer to the human V region germline sequence (i.e., before somatic hypermutation) than to sequences from other species, using the Immunogenetics Information DomainGapAligntool (Ehrenmann F, Kaas Q and Lefranc MP 2010, supra) for evaluation.

[0161] Table 1 summarizes the best aligned V genes and alleles, including species, IMGT V gene and allele names, and domain tags, as well as the percent identity and overlap (number of aligned amino acids assigned to the V region) between the humanized variants and the IMGT amino acid reference sequence, from which the WHO INN predictions for the humanized variants were obtained. Table 1: Antibody INNs assigned by WHO for humanized variants

[0162] 1.2.2 Humanized anti-IGLV3-21 R110 Antibody expression

[0163] Subsequently, the amino acid sequence of the humanized variant produced by the fusion antibody was converted into a nucleotide sequence using Geneiouse software (Geneious Prime 2, Auckland, New Zealand). The VH sequence was fused to the human IgG1 isotype constant domain sequence (SEQ ID NO: 50 represents IgG1 constant nucleotides) by PCR, and the VL sequence was fused to the human IgK isotype constant domain (SEQ ID NO: 51 represents IgK constant nucleotides) to produce humanized heavy and light chains, and the antibody gene sequence was transiently expressed in Chinese hamster ovary cells (CHO). The obtained cell culture supernatant containing the antibody was clarified by centrifugation and filtration. The humanized antibody was purified from the cell culture supernatant by affinity chromatography. As determined by reduction and denaturation sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purity of the antibody was determined to be >95%. The protein content and concentration of the antibody were analyzed by size exclusion chromatography (SEC) in PBS buffer. All steps were carried out using the most advanced equipment and technology.

[0164] This approach generated eight humanized antibodies: HC6-LC6, HC7-LC6, HC6-LC7, HC7-LC7, HC6-LC8, HC7-LC8, HC6-LC9, and HC7-LC9.

[0165] 1.2.3 Humanized anti-IGLV3-21 R110 Antibody binding affinity

[0166] After expressing the eight humanized antibodies HC6-LC6, HC7-LC6, HC6-LC7, HC7-LC7, HC6-LC8, HC7-LC8, HC6-LC9, and HC7-LC9, soluble forms of IGLV3-21 were used. R110 BCR binding assays confirmed the high affinity of these antibodies, as none of them showed binding greater than about 3·10 -10 The value of M.

[0167] In summary, in order to define the relationship between these antibodies and IGLV3-21 R110The binding affinity of the B cell receptor of the human IgM protein was determined by transiently expressing a soluble recombinant version of the BCR (170.5 kDa according to the sequence of SEQ ID NO:52 for HC and SEQ ID NO:53 for LC) in a 293-HEK cell line using the protocol described in Section 1.1 of this example, and monitoring its binding to immobilized anti-IGLV3-21 by bio-layer interferometry (BLI) on a Fortebio Octet instrument (Satorius). R110 Binding of antibodies.

[0168] First, humanized anti-IGLV3-21 was captured by an indirect capture reagent, anti-human IgG Fc antibody. R110 Antibodies were immobilized on the biosensor and kinetic measurements were performed. Anti-IGLV3-21 was immobilized on the biosensor at a concentration of 0.01875 μg / ml. R110 Antibody loading to produce capture levels between 0.30 and 0.34 nm for anti-IGLV3-21 R110 Antibodies. A 9 nM BCR fragment solution was prepared in running buffer (PBS, 0.02% Tween 20, 0.1% BSA, 0.05% sodium azide) and serially diluted 1:3 to obtain 7 concentrations from 9 to 0.012 nM (9 nM, 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, and 0.012 nM). Then, anti-IGLV3-21 R110 The antibody-captured biosensors were immersed in wells containing different concentrations of soluble BCR fragments for 900 s (binding phase), followed by a 1200 s dissociation step in running buffer. These steps were performed at a constant shaking speed of 1000 rpm. All reagents were used as described by the manufacturer. Sensorgrams were generated after double reference calibration (buffer and blank sensor) to compensate for the capture of anti-IGLV3-21 R110 The natural dissociation of antibodies and the nonspecific binding of soluble BCR fragments to the sensor surface. a ) and the dissociation rate constant (k d ) ratio, and the dissociation rate constant (K) was obtained by fitting the sensorgram to a first-order 1:1 binding model using Fortebio data analysis software (Satorius). D Table 2: Comparison of humanized antibody variants with soluble IGLV3-21 measured by Fortebio R110 B cell receptor monovalent K D Value and anti-IGLV3-21 R110 Antibody capture level Humanized Antibodies KD(M) Capture Level(nm) HC6-LC6 2.11E-10 0,305 HC7-LC6 1.75E-10 0,322 HC6-LC7 3.47E-10 0.337 HC7-LC7 2.69E-10 0.323 HC6-LC8 2.77E-10 0.313 HC7-LC8 2.49E-10 0.330 HC6-LC9 2.91E-10 0.311 HC7-LC9 3.13E-10 0.280

[0169] 2. Humanized anti-IGLV3-21 R110 CAR

[0170] 2.1 Humanized anti-IGLV3-21 R110 Generation of CAR

[0171] Subsequently, a structural model of a humanized single-chain variable fragment (scFv) comprising the amino acid sequences of humanized LC6 and HC7 variants fused to a linker (Li, SEQ ID NO: 46) was predicted ( Suit, Inc.NY, USA). The obtained sequence is required to be used as a template for designing the remaining single-stranded fragments.

[0172] The amino acid sequence of the humanized scFv was converted to nucleotide sequence using Geneiouse software (Geneious Prime 2, Auckland, New Zealand), and two different CAR constructs were used to design humanized anti-IGLV3-21 from scFv. R110 CAR:

[0173] CAR construct 1 includes a spacer derived from a human IgG4 hinge (UniProtKB P01861IGHG4_HUMAN). The co-stimulatory domain is derived from the CD28 transmembrane domain (UniProtKB P10747 CD28_HUMAN) and the human receptor 4-1BB (UniProtKB Q07011TNR9_HUMAN). CD3ζ (UniProtKB P20963 CD3Z_HUMAN) is used as a signaling domain.

[0174] CAR Construct 2 differs from CAR Construct 1 in that it contains an additional spacer sequence between the IgG4 hinge and the CD28 sequence, which is derived from the CH2-CH3 domain of IgG4 (UniProtKB P01861 IGHG4_HUMAN).

[0175] The obtained humanized anti-IGLV3-21 R110 The antigen binding domain sequences of CARs are described in Table 3. Table 3: Humanized anti-IGLV3-21 R110 CAR, antigen binding domain sequence:

[0176] 2.2. T cell epitope screening

[0177] Presentation of peptide sequences in the groove of MHC class II molecules leads to activation of CD4+ T cells and an immunogenic response. In order to reduce this response, therapeutic proteins can be designed to avoid incorporating T cell epitopes that can activate T cells by reducing the affinity of binding to MHC class II molecules.

[0178] To demonstrate resistance to IGLV3-21 R110The antigen binding domain of CAR exhibits reduced immunogenicity compared to the mouse sequence, and the humanized VL and VH protein sequences of the binding domain and those of the mouse antibody mAB01-01 are screened for MHC class II binding peptides. Screening is performed using SMM-align known in the art (Wang P, Sidney J, Kim Y, et al. 2010. Peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics. 11: 568; Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 8: 238). The following eight alleles representing more than 99% of the world's population were used as the standard allele set for predicting MHC class II epitopes: DRB1*01:01; DRB1*03:01; DRB1*04:01; DRB1*07:01; DRB1*08:02; DRB1*11:01; DRB1*13:02; DRB1*15:01 (Gonzalez-Galarza FF, Christmas S, Middleton D and Jones AR 2011. Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acid Research, 39, D913-D919; Greenbaum J, Sidney J, Chung J, et al. 2011 Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics 63(6):325-35).

[0179] Tables 4 and 5 show the results of the VL and VH screening, with high affinity T cell epitope cores shown in bold (IC50 < 50 nM).

[0180] The results of the VL screen are shown in Table 4. The human germline sequence IGKV1-9 was also analyzed for comparison, and any potential T cell epitopes present in this germline sequence and matched in the humanized variants are shown in italics. Table 4: MHC class II epitope predictions for light chain variants

[0181] The results of the VH screen are shown in Table 5. The human germline sequence IGHV4-59 was also analyzed for comparison, and any potential T cell epitopes present in this germline sequence and matched in the humanized variants are shown in italics. Table 5: MHC class II epitope predictions for heavy chain variants

[0182] As can be readily seen from Tables 4 and 5, the murine VH contains a T cell epitope within the framework 1 region of the sequence, which is absent in the humanized variants (HC6, HC7).

[0183] Example 2

[0184] Generation of primary human T cells expressing CAR

[0185] In order to explore the humanized anti-IGLV3-21 R110 CAR redirects immune cell specificity and responsiveness to IGLV3-21 R110 The ability of positive BCR to generate T cells expressing humanized anti-IGLV3-21 by viral vector transduction R110 CAR H1 and H2 T cells are produced using techniques known in the art and documented (e.g., Yang S, Zhou X, Li R, Fu X, Sun P. Optimized Pei-Based Transfection Method for Transient Transfection and Lentiviral Production. Curr Protoc Chem Biol (2017) 9 (3): 147-57. Epub 2017 / 09 / 15. doi: 10.1002 / cpch.25). In order to test T cells in different states, healthy people and CLL patients with active CLL (CLL425; CLL433) and remission CLL (CLL453) were used as T cell donors.

[0186] Production of CAR gene delivery vector particles (lentiviral vector particles)

[0187] For the expression of scFv in CAR construct 1, vector pJ2459_19loBB (SEQ ID NO: 48) was used, while vector pJ2487_19loBB (SEQ ID NO: 49) was used to express scFv in CAR construct 2. R110 Two vectors of CAR.

[0188] According to the manufacturer's instructions, use In- The ScFv sequence was cloned into two CAR vectors pJ2487_19loBB and pJ2459_19loBB using the cloning kit (Takara Biosciences, Cat.639650) through NheI (New England Biolabs, Cat.R3131M) and RsrII (New England Biolabs, Cat.R0501S) restriction sites.

[0189] Lentiviral vector particles were produced in HEK293T cells (DSMZ-German Collection of Microorganisms and CellCultures GmbH, Cat. ACC 635). 5×10 6 HEK293T cells were seeded into 25 cm 2 DMEM GlutaMAX supplemented with 10% (v / v) FBS (Life Technologies, Cat. 10500064) and 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. 15140122) in the culture flask TM (ThermoFisher, Cat. 31966021) and incubated at 37°C in an atmosphere containing 5% CO 2 After 18-24 hours, the medium was replaced with 10 mL of fresh medium containing 25 μM chloroquine (Sigma, Cat. C6628). 215 μg CAR construction vector was mixed with 10 μg gag / pol plasmid pMDLg / pRRE (Addgene, Cat.12251), 5 μg rev plasmid pRSV-Rev (Addgene, Cat.12253) and 2 μg envelope plasmid pCMV-VSV-G (Addgene, Cat.8454) in (Sigma, Cat.C1016). 500 μL of 2x HEPES buffered saline (Thermo Fisher Cat.15488749) was added to the DNA mixture, incubated at room temperature for 20 minutes and added to the cells. After 24, 48 and 72 hours, the cell culture medium was collected, filtered through a 0.45 μm injection filter (Sarstedt, Cat.83.1826) and stored at -80 ° C until transduction.

[0190] T cell isolation and transduction

[0191] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors or CLL patients and collected in K3 ethylenediaminetetraacetic acid (EDTA) S-Monovetten (EDTA tubes, Sarstedt AG, Cat. 02.1066.001) EDTA tubes. Whole blood was layered into lymphocyte separation medium (Anprotec, Cat. AC-AF-0018) and centrifuged at 500 x g for 30 min without stopping. PBMCs were harvested in the interphase and washed with phosphate-buffered saline (PBS). In the case of CLL samples only, cells were isolated in Miltenyi according to the manufacturer's instructions. PBMCs were depleted of B cells using the Human B Cell Isolation Kit II (Miltenyi, Cat. 130-091-151) according to the manufacturer's instructions. T cells were isolated based on negative selection using the Pan T cell isolation kit (Miltenyi, Cat. 130-096-535) on the instrument.

[0192] The cells were cultured at 37°C in a humidified atmosphere including 5% CO2 in RPMI 1640 medium supplemented with 10% (v / v) FBS (Life Technologies, Cat. 10500064), 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. 15140122), 50 μM β-mercaptoethanol (Thermo Fisher, Cat. 31350010), and GlutaMAX. TMSupplement (Thermo Fisher, Cat. 61870044), all T cells were expanded, and 50 U / mL IL-2 (StemCell, Cat. 78036.1) was added every 48 hours (CTL medium). 1 Mio T cells were mixed with washed CD3 / CD28 beads (ImmunoCult TM Human CD3 / CD28 T cell activator, StemCell, Cat.10971) was seeded in 2 mL CTL medium in each well of a 24-well plate at a 1:1 bead:T cell ratio and 50 U / mL IL-2 (StemCell, Cat.78036.1). After 24 hours, 1.3 mL of medium was removed from each well, 5 μg / mL polybrene (Merck, Cat.TR-1003-G) and lentiviral particles were added, and centrifuged continuously at 800g and 32°C for 45 minutes. The cells were incubated at 37°C for 4 hours and fresh CTL medium was added. On the second day and every two days thereafter, half of the medium was replaced and fresh IL-2 was added. After 6 days, the CD3 / CD28 beads were removed. Cells were expanded for up to 21 days. Anti-EGFR antibody cetuximab was used on a BD FACSCalibur TM On the instrument, check the transduction efficiency by flow cytometry. Cells with E cells above the threshold of the unstained control in the fluorescent channel FL1 are considered to be successfully transduced. A transduction efficiency above 80% is acceptable.

[0193] The anti-IGLV3-21 produced is described in Table 6 R110 CAR-T cells. Table 6 CAR-T cells scFv T cell source Carrier CARHDh1 H1 Healthy donor pJ2459_19loBB CARHDh2 H2 Healthy donor pJ2459_19loBB CAR87HDh1 H1 Healthy donor pJ2487_19loBB CAR87HDh2 H2 Healthy donor pJ2487_19loBB CLL425CARCLLh1 H1 CLL patients 425 pJ2459_19loBB CLL425CARCLLh2 H2 CLL patients 425 pJ2459_19loBB CLL433CARCLLh1 H1 CLL patients 433 pJ2459_19loBB CLL433CARCLLh2 H2 CLL patients 433 pJ2459_19loBB CLL453CARCLLh1 H1 CLL patients 453 pJ2459_19loBB CLL453CARCLLh2 H2 CLL patients 453 pJ2459_19loBB

[0194] Example 3

[0195] Humanized anti-IGLV3-21 R110 In vitro cytotoxicity assay of CAR-T cells

[0196] A. Provide target cells and effector cells

[0197] a) Target cells:

[0198] CAR efficacy was tested using OCI-LY1 wt, OCI-LY1 R110, primary CLL PBMCs, or healthy B cells.

[0199] a.1) OCI-LY1 wt cell line (OCI wt)

[0200] The OCI-LY1 (wild type) wt cell line, derived from the bone marrow of a 44-year-old male with B-cell non-Hodgkin's lymphoma, was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Cat. ACC722).

[0201] a.2) OCI-LY1 R110 cell line (OCIR110)

[0202] Neo-epitope (i.e., cancer-specific peptide) engineered cell line OCI-LY1 R110 (which expresses IGLV3-21 R110 Light chain) as part of the BCR with an induced R110 point mutation, produced by OCI-LY1 wt cells. First, at 37°C in an atmosphere containing 5% CO 2 The cells were cultured in a humidified environment in RPMI 1640 medium supplemented with 20% (v / v) FBS (Life Technologies, Cat. 10500064) and 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. 15140122). TM OCI-LY1 wt cells were cultured in 4% paraformaldehyde (PAGE) supplement (Thermo Fisher, Cat. 61870044). R110The light chain was used as part of a hybrid BCR, and the coding sequence (SEQ ID NO: 47) was cloned into the lentiviral gene ontology (LeGO) vector LeGO-iC2-Puro+ (Addgene, Cat. 27345) by AsiSI (New England Biolabs, Cat. R0630S) / EcoRI (New England Biolabs, Cat. R3101L). Lentiviral particles were generated by HEK293T cells (DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Cat. ACC 635) according to the manufacturer's protocol and used to transduce OCI-LY1 wt cells. As indicated in the related Example 2, both the production of lentiviral particles and cell transduction are well-known methods and are known to experts (see, for example, Yang S, et al. (2017) supra; Weber K, Bartsch U, Stocking C, Fehse BA Multicolor Panel of Novel Lentiviral "Gene Ontology" (Lego) Vectors for Functional Gene Analysis. Molecular Therapy (2008) 16 (4): 698-706.). Using APC anti-IGLV3-21 R110 Antibody (AVA LifeScience, Cat. AVA-D01-APC / 01P0250), confirmed by flow cytometry IGLV3-21 R110 Expression of light chain. OCI-LY1 R110 cells were cultured as described for OCI-LY1 wt.

[0203] a.3) Primary CLL PBMC

[0204] Blood samples were collected from CLL patients with R110 point mutations (CLL425; CLL438; CLL442) and from patients with different types of CLL (CLL399; CLL426; CLL427) with wt IGLV3-21. G110 Primary CLL PBMCs were isolated from the blood of CLL patients with light chains and collected in EDTA tubes. Whole blood was layered into lymphocyte separation medium (Anprotec, Cat. AC-AF-0018) and centrifuged at 500 x g for 30 minutes without interruption. PBMCs were harvested in the interphase and washed with phosphate buffered saline (PBS).

[0205] a.4) Healthy B cells: primary polyclonal B cells (B HD )

[0206] PBMCs were isolated from blood of healthy donors and collected in EDTA tubes. Whole blood was layered into lymphocyte separation medium (Anprotec, Cat. AC-AF-0018) and centrifuged at 500 x g for 30 minutes without interruption. PBMCs were harvested during the interphase and washed with phosphate buffered saline (PBS). The cells were centrifuged in a Miltenyi ELISA kit according to the manufacturer's instructions. Primary polyclonal non-malignant B cells were isolated using the Human B Cell Isolation Kit II (Miltenyi, Cat. 130-091-151) on the instrument.

[0207] b) Effector cells:

[0208] b.1) Anti-IGLV3-21 R110 CAR T cells

[0209] Use patients from healthy donors, patients with active CLL (CLL425; CLL433) or patients expressing humanized anti-IGLV3-21 R110 CAR T cells prepared according to Example 2 for CLL patients with remission CLL (CLL453).

[0210] b.2) Healthy donors and CLL patients without transduced CAR T cells

[0211] Untransduced T cells from healthy donors (CARHDUTD, control) and from CLL patients (eg, CLL433 CARCLLUTD; CLL453 CARCLLUTD), isolated according to Example 2, were used to reduce background.

[0212] b.3) Anti-human thyroid stimulating hormone receptor (TSHR) CAR T cells (CARHDTSHR)

[0213] CAR T cells directed to human thyroid stimulating hormone receptor from healthy donors were used as controls. In order to generate anti-TSHR CAR T cells, the paratope sequence of autoantibodies targeting human thyroid stimulating hormone receptor (TSHR) was obtained from the literature (Rees Smith, B., Sanders, J., Evans, M., Tagami, T. & Furmaniak, J. TSH receptor-autoantibody interactions. Horm Metab Res 41, 448-455 (2009)), translated into scFv format, and cloned into pJ2459 lentiviral vector.

[0214] b.4) Anti-CD19 CAR T cells

[0215] As previously published by Hudecek, M. et al. (The nonsignaling extracellular spacerdomain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 3, 125-135 (2015)), CAR targeting pJ2459-CD19 was used to equip T cells from healthy donors (CARHDCD19) and CLL patients (e.g. CLL433CARCLLCD19; CLL453CARCLLCD19) by lentiviral transduction.

[0216] B. Cytotoxicity Assay

[0217] 2×10 4 Target cells seeded at a density of 10 cells / well in 96-well plates were co-cultured with effector cells at an effector cell to target cell (E:T) ratio of 5:1 in complete CTL medium consisting of RPMI 1640 medium supplemented with 10% (v / v) FBS (Life Technologies, Cat. 10500064), 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. 15140122), 50 μM β-mercaptoethanol (Thermo Fisher, Cat. 31350010), and 50 U / mL IL-2 (StemCell, Cat. 78036.1). TM Supplement (Thermo Fisher, Cat. 61870044). Caspase-3 / 7 green apoptosis reagent (Sartorius, Cat. 4440) was used to assess CAR T cell-mediated tumor cell killing. Read-out of dead (=green fluorescent) target cells was performed on S3 (Sartorius). Software (version 2022A) calculates killing graphs. Total fluorescence of each well and time point is measured relative to the control wells and recorded or plotted over time in a timely manner after a specific time point. Target cells are used in combination with untransduced T cells as controls to set the background. Additional control assays are performed using effector cells only or using target cells only. All values ​​are obtained by repeating three times.

[0218] From Figure 3 (which shows representative images of cytotoxicity assays) and Figures 4, 6, Figure 7 As can be easily seen from the killing graph shown in Figure 9, unlike anti-CD19 CAR T cells, humanized anti-IGLV3-21 R110 scFv sequence-expressing CAR T cells selectively kill IGLV3-21 R110 Cell lines and primary CLL cells.

[0219] The cytotoxicity assay also revealed that the killing ability of scFv H1 and scFv H2 was significantly higher in CAR construct 1 than in CAR construct 2, apparently this may be due to the shorter spacer between the binding domain and the transmembrane domain of CAR construct 1.

[0220] As depicted in Figure 4, monitoring over time showed that anti-IGLV3-21 from healthy human donors R110 CAR T cells specifically kill cells expressing IGLV3-21 R110 Cell line. IGLV3-21 R110 Negative cell lines were not affected by these CAR T cells.

[0221] Untransduced control CAR T cells prepared from healthy donors or CLL patients neither killed IGLV3-21 R110 Positive cell lines, but not IGLV3-21 R110 Negative cell lines (Figure 4, Figure 6 and Figure 9). Likewise, both cell lines were not affected by anti-TSHR CAR T cells derived from healthy donors (Figure 4, Figure 6, Figure 9).

[0222] The cytotoxicity assay also demonstrated that humanized anti-IGLV3-21 derived from healthy human donors R110 scFv sequence-expressing CAR T cells selectively kill freshly prepared IGLV3-21 R110 Positive CLL cells (Figure 6). Isolated primary polyclonal non-malignant B cells were also unaffected by these cells, suggesting that epitope-specific targeting preserves normal B cells ( Figure 7 In contrast, anti-CD19 CAR T cells killed all B cells tested equally, regardless of the presence of IGLV3-21. R110 epitopes (e.g., Figures 6 and Figure 7 ).

[0223] To extend the study to expressing IGLV3-21 R110Additional cytotoxicity assays were performed using thawed CLL samples. Although the background in these experiments was generally high due to the low viability of primary CLL cells, it was confirmed that IGLV3-21 R110 Specific killing of positive CLL cells.

[0224] In addition, CAR T cells in the blood of CLL patients showed selective killing of IGLV3-21 R110 The ability of positive cells, despite the well-recognized impaired T cell functionality in this disease, can be representatively seen in Figure 9, which shows killing graphs generated using CAR T cells from two CLL patients, one with active CLL and one with CLL in remission.

[0225] Example 4

[0226] Humanized anti-IGLV3-21 in the presence of BTK inhibitors R110 Comparative cytotoxicity of CAR-T cells Determination

[0227] Co-administration of BTK inhibitors (such as ibrutinib, a BTK / ITK inhibitor known to restore T cell functionality) to CAR T cells may produce additional anti-tumor effects. To test this in the R110CAR T setting, first, the toxicity of ibrutinib to OCI-Ly1 target cells was evaluated. To this end, 2×10 4 OCI-Ly1 wt cells and OCI-Ly1_R110 cells were seeded in 200 μl of culture medium supplemented with serial dilutions of ibrutinib (PCI-32765, Selleck Chemicals, Cat. S2680) ranging from 0.01 to 20 μM. Cells grown under the same conditions without ibrutinib served as controls. After 24 h of incubation, viable cells were counted (trypan blue exclusion method) using the Vi-CELL XR system (Beckman Coulter). Then, the percentage of cell growth was plotted according to the total number of cells treated with ibrutinib relative to the total number of untreated cells ( Fig.10 ). This analysis revealed that 0.08 μM ibrutinib as the highest dose did not result in a decrease in target cell viability. Therefore, this dose was selected for further testing of CAR T efficacy.

[0228] According to Example 3, cells from healthy donors and IGLV3-21 R110 Humanized anti-IGLV3-21 for positive CCL patient (CLL425) R110 Comparative cytotoxicity assay of CAR-T cells. For killing assays, at the time of inoculation of target and effector cells (day 0), 0.08 μM ibrutinib was added per well for the duration of the assay (16 h).

[0229] like Fig.11 As shown, the addition of ibrutinib at a subtherapeutic dose increased the killing capacity of CLL-derived CAR T cells to the level of CAR-T cells derived from healthy donors.

[0230] Example 5

[0231] Interferon-γ (IFNγ) Overview

[0232] The immune effector cytokine IFN-γ was examined during the cytotoxicity assay. Following co-culture of effector cells with target cells, the bead-based immunoassay technology LEGENDplex was used according to the manufacturer's protocol. TM Human B Cell Panel Standard (BioLegend, Cat. 740537) measures the concentration of IFN-γ in the culture supernatant from 8 to 16 hours or 36 hours. 25uL of supernatant is used for each well. TM Fluorescence was measured on the instrument. Values ​​below the detection limit were considered zero.

[0233] like Figure 5A , Fig. 8A and Fig.12 As shown, consistent with the cytotoxicity assay results, elevated IFNγ levels were measurable in the corresponding supernatants of cell killing shown by CAR T cells.

[0234] Example 6

[0235] Overview of Interleukin 6 (IL-6)

[0236] The immune effector cytokine IL-6 was examined in a cytotoxicity assay. Following co-culture of effector cells with target cells, the bead-based immunoassay technology LEGENDplex was used according to the manufacturer's protocol. TM The concentration of IL-6 in the supernatant of the 8- to 36-hour culture was measured using a human B cell panel standard (BioLegend, Cat. 740537). 25 uL of supernatant was used per well. TM Fluorescence was measured on the instrument. Values ​​below the detection limit were considered zero.

[0237] like Figure 5B and Figure 8B As shown, consistent with the cytotoxicity assay results, elevated IL-6 levels were measurable in the corresponding supernatants of cell killing shown by CAR T cells.

Claims

1. Humanized chimeric antigen receptor (CAR), comprising humanized IGLV3-21 R110 Binding domain, transmembrane domain and cytoplasmic domain, wherein the humanized IGLV3-21 R110 The binding domain includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region (VL) includes a light chain complementary determining region 1 (L-CDR1) having the amino acid sequence shown in SEQ ID NO:1, a light chain complementary determining region 2 (L-CDR2) having the amino acid sequence shown in SEQ ID NO:2, and a light chain complementary determining region 3 (L-CDR3) having the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region (VH) includes a heavy chain complementary determining region 1 (H-CDR1) having the amino acid sequence shown in SEQ ID NO:4, a heavy chain complementary determining region 2 (H-CDR2) having the amino acid sequence shown in SEQ ID NO:5, and a heavy chain complementary determining region 3 (H-CDR3) having the amino acid sequence shown in SEQ ID NO:

6.

2. The CAR according to claim 1, characterized in that: Any combination of a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:

12.

3. The CAR according to claim 2, characterized in that: A VL having the amino acid sequence shown in SEQ ID NO:7 and a VH having the amino acid sequence shown in SEQ ID NO:11, or a VL having the amino acid sequence shown in SEQ ID NO:7 and a VH having the amino acid sequence shown in SEQ ID NO:

12.

4. The CAR according to any one of the preceding claims, characterized in that A VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity to the amino acid sequence of the VL according to claim 3, and The amino acid sequence of the VH according to claim 3 has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity.

5. The CAR according to any one of the preceding claims, wherein the humanized anti-IGLV3-21 R110 The binding domain is a scFv.

6. The CAR of claim 5, wherein the scFv has an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:

20.

7. A polynucleotide encoding the CAR according to any one of the preceding claims. A vector comprising the polynucleotide according to claim 7.

9. A cell expressing the CAR according to any one of claims 1-6.

10. The cell of claim 9, wherein the cell is a T cell.

11. A pharmaceutical composition comprising the CAR according to any one of claims 1 to 6, the polynucleotide according to claim 7, the vector according to claim 8 or the cell according to claim 9 or 10, and a pharmaceutically acceptable carrier.

12. Use of the CAR according to any one of claims 1 to 6, the polynucleotide according to claim 7, the vector according to claim 8, the cell according to claim 9 or 10, or the pharmaceutical composition according to claim 11 in treating a disease.

13. A method for treating a disease, comprising the step of administering to a subject in need thereof a CAR according to any one of claims 1 to 6, a polynucleotide according to claim 7, a vector according to claim 8, a cell according to claim 9 or 10, or a pharmaceutical composition according to claim 11.

14. The method according to claim 13, comprising the steps of: i) providing immune cell populations, ii) introducing the polynucleotide according to claim 7 or the vector according to claim 8 into the immune cell, iii) culturing the immune cells under conditions that allow expression of the CAR, and iv) administering said cells from step (iii) to a subject.

15. Use of the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to claim 12, or the method according to claim 13 or 14, wherein the disease is chronic lymphocytic leukemia (CLL).

16. Use of the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to claim 12 or 15, or the method according to any one of claims 13 to 15, wherein the CAR, the polypeptide, the vector, the cell or the pharmaceutical composition is to be administered in combination with an additional therapeutic agent.

17. The CAR, the polypeptide, the vector, the cell or the pharmaceutical composition according to claim 16, wherein the additional therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor.

18. A kit comprising the pharmaceutical composition according to claim 11.

Images