RU2839662C1 - T-cells with two chimeric antigen receptors (car) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, namely to a genetically engineered T-cell expressing on its surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22), and a chimeric antigen receptor specific for CD20 (CAR20), a method for production thereof, as well as a pharmaceutical composition containing it. Also disclosed is an isolated polynucleotide encoding CAR20 and CAR22, as well as a vector containing said polynucleotide.

EFFECT: invention is effective for treating cancer associated with CD22 expression, for treating cancer associated with CD20 expression, as well as for treating cancer associated with CD20 and CD22 expression.

17 cl, 8 dwg, 7 tbl, 9 ex

Description

Field of technology

The present invention relates to the field of cellular immunotherapy and, more specifically, to novel engineered immune cells expressing two CARs directed against two different targets and useful for the treatment of cancer.

Prior art

Approximately every three minutes, one person in the United States is diagnosed with a blood cancer. Previous projections for 2020 were for 178,520 new cases of leukemia, lymphoma, and myeloma. In the United States, new cases of leukemia, lymphoma, and myeloma were expected to account for 9.9% of the expected 1,806,590 new cancer cases diagnosed in 2020 (Cancer Facts & Figures, 2020. American Cancer Society).

The development of chimeric antigen receptor (CAR) T-cell therapy for hematological malignancies represents one of the most significant therapeutic advances of the past decade (Holstein et al., Clin. Pharmacol. Ther., 2020, 107(1): 112–122). Indeed, as a rapidly evolving field of oncology, adoptive transfer of CAR T cells has shown remarkable efficacy in the treatment of hematological malignancies, as reported in a number of clinical trials.

Chimeric antigen receptor (CAR)-expressing immune cells are cells that are genetically engineered to express CARs and are typically designed to recognize specific tumor antigens and kill cancer cells that express those tumor antigens. It is possible that CAR-expressing immune cells can activate the immune system to eliminate tumors. Typically, these are T cells that express CAR receptors (“CAR-T cells”), or natural killer (NK) cells that express CARs (“CAR-NK cells”), or macrophages that express CARs.

CARs are synthetic receptors consisting of a targeting moiety linked to one or more signaling domains in a single fusion molecule. Typically, the binding portion of a CAR consists of an antigen-binding domain derived from a monoclonal antibody consisting of a single-chain variable fragment (scFv) that contains the light and heavy variable fragments of the monoclonal antibody connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domains for first-generation CARs are derived from the cytoplasmic region of CD3zeta or the gamma chains of the Fc receptor. First-generation CARs have been shown to successfully redirect T-cell cytotoxicity, but they fail to provide prolonged spreading and antitumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and proliferation of CAR-modified T cells. CARs have successfully enabled T cells to be redirected against antigens expressed on the surface of tumor cells in a variety of malignancies, including lymphomas and solid tumors (Jena, Dotti et al., Blood, 2010, 116(7): 1035-1044).

Adoptive immunotherapy, which involves the transfer of autologous or allogeneic antigen-specific T cells generated ex vivo, is a promising strategy for the treatment of viral infections and cancer, as evidenced by the increasing number of CAR-T cells approved by the US Food and Drug Administration (FDA) (e.g., Novartis' anti-CD19 CAR-T tisagenlecleucel (Kymriah™) for the treatment of pre-B-cell acute lymphoblastic leukemia, Kite Pharma's anti-CD19 CAR-T axicabtagene ciloleucel (Yescarta™) for the treatment of certain types of large B-cell lymphoma in adults).

Despite advances in CAR-T cell therapy research and development, there remains a need for improved CAR-T cells that can target a broader range of cancers, as well as recurrent cancers and/or cancers for which the distribution of cancer-associated antigens is highly variable and evolves over time, during or after treatment.

Brief description of the invention

The present invention provides novel CAR-T cells targeting CD20 and CD22 antigens that can be activated by tumor cells expressing CD20 and CD22 at various levels and are an improvement over prior art CAR-T cells.

The first object refers to genetically engineered immune cells expressing on their cell surface a chimeric antigen receptor (CAR) specific to CD22 (CAR22) and a chimeric antigen receptor specific to CD20 (CAR20):

a) where the said CAR22 contains:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

b) where the said CAR20 contains:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In one of the objects, the genetically engineered immune cells are TCR-negative T cells.

Another object of the present invention relates to a pharmaceutical composition,

containing the specified engineered immune cells or population of cells,

containing said engineered immune cells and a pharmaceutically acceptable excipient.

Another object relates to an isolated polynucleotide comprising: a) a nucleic acid encoding CAR22, comprising: i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

b) nucleic acid encoding CAR20, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

Other objects relate to a vector containing said polynucleotides, as well as to a host cell containing said polynucleotides or vector.

Another object relates to a method for obtaining said engineered immune cells.

Other objects relate to the said engineered immune cells or cell populations for use as a medicinal product.

Another object relates to said engineered immune cells for use in the treatment of cancer or inflammatory disease, in particular cancer or inflammatory disease associated with the expression of CD20 and/or CD22.

Brief description of the figures

Figure 1. Detection of CD20×CD22 or CD22×CD20 CAR. Flow cytometric analysis showing detection of CD20 CAR or CD22 CAR in untransduced (UT) T cells or cells transduced with CD22, CD22×CD20, or CD20×CD22 CAR constructs using the protocol described in the example.

Figure 2. Flow cytometry analysis of different Raji cells obtained after CD20 and CD22 TALEN® treatment and cell sorting.

Figure 3. Percent lysis of CD20×CD22, CD22×CD20 and CD22 CAR-T cells from 2 different donors relative to Raji cells expressing or not expressing CD20 and/or CD22 antigens.

Figure 4. Percent lysis of CD20×CD22, CD22×CD20, and CD22 CAR-T cells over time relative to Raji cells expressing CD20 and CD22 (A), expressing CD22 only (B), or expressing CD20 only (C).

Figure 5. Dose-dependent in vivo control of tumor burden following treatment with the indicated dose of CD20xCD22, CD22xCD20, and CD22 CAR T cells measured by bioluminescence.

Figure 6. Survival curve of animals treated with the indicated doses of CD20xCD22, CD22xCD20, and CD22 CAR-T cells in a disseminated B-cell lymphoma model.

Figure 7. Survival curve of animals treated with the indicated doses of CD20xCD22, CD22xCD20, and CD22 CAR-T cells in a subcutaneous B-cell lymphoma model.

Figure 8. Release of gamma interferon by CD20×CD22, CD22×CD20, CD20 and CD22 CAR-T cells on Daudi cells.

Corresponding designations used in the description and examples: Anti-CD20 CAR=CAR20=CD20CAR Anti-CD22 CAR=CAR22=CD22CAR

Anti-CD20 CAR/aHTH-CD22 CAR=CAR20x22 or CAR22x20=CD20xCD22CAR or CD22xCD20CAR.

Detailed description of the invention

Unless otherwise defined in this description, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the art of gene therapy, biochemistry, genetics and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention with the corresponding methods and materials described herein. The disclosures of all publications, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety. In case of conflict, the present description, including definitions, controls. In addition, the materials, methods, and examples are provided by way of illustration only and do not limit the scope of the present invention unless otherwise specifically indicated.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully described in the literature. See, for example, the book: "Chemp Protocols in Molecular Biology", Frederick M. Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Sambrook et al., the book: "Molecular Cloning: A Laboratory Manual", 3rd ed., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); the book: Oligonucleotide Synthesis", edited by M. J. Gait, 1984; US 4683195; the book: "Nucleic Acid Hybridization"), edited by B. D. Harries and S. J. Higgins, 1984; book: «Transcription And Translation)) edited by B. D. Hames and S. J. Higgins, 1984; book: «Culture Of Animal Cells», R. I. Freshney, Alan R. Liss, Inc., 1987; book: «Lnmobilized Cells And Enzymes», IRL Press, 1986; B. Perbal, «A Practical Guide To Molecular Cloning))), 1984; series «Methods In ENZYMOLOGY», edited by J. Abelson and M. Simon, Academic Press, Inc., New York, especially volumes 154 and 155 (edited by Wu et al.) and volume 185 «Gene Expression Technology)) edited by D. Goeddel; book: «Gene Transfer Vectors For Mammalian Cells)», edited by J. H. Miller and M. P. Calos, 1987, Cold Spring Harbor Laboratory; book: «Immunochemical Methods In Cell And Molecular Biology» edited by Mayer and Walker, Academic Press, London, 1987; book: «Handbook Of Experimental Immunology)», volumes I-IV, edited by D. M. Weir and C.C. Blackwell, 1986; book: «Manipulating the Mouse Embryo», edited by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986.

Definitions

As used herein, the term "antigen associated with a disease state" refers to an antigen present or overexpressed in a disease. When the antigen is CD20 or CD22, the terms "CD20-associated disease" or "CD22-associated disease" refer to a disease like cancer or an inflammatory disease in which the CD20 or CD22 antigen is typically present in tumor cells or cells that provoke an inflammatory response (particularly B cells). The antigen associated with a disease state that is cancer, i.e., the "cancer-associated antigen", may be a tumor antigen as described herein.

As used herein, the term "CD20" refers to an antigenic determinant known to be found on B cells. Human CD20 is also referred to as membrane-spanning 4-domains, subfamily A, member 1 (MS4A1). The amino acid and nucleic acid sequences of human and mouse CD20 can be found in publicly available databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be accessed under the accession numbers NP 690605.1 and NP 068769.2, and the nucleic acid sequence encoding human CD20 transcript variants 1 and 3 can be accessed under the accession numbers NM 152866.2 and NM 021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen in the extracellular domain of the CD20 protein. In another aspect, the CD20 protein is expressed on a cancer cell. As used herein, the term "CD20" includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants, of the full-length wild-type CD20 sequence.

As used herein, the term "CD22" refers to an antigenic determinant known to be found on leukemia progenitor cells. The amino acid and nucleic acid sequences of human and mouse can be found in publicly available databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1-5 can be found under the accession numbers NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleic acid sequences encoding human CD22 variants 1-5 can be found under the accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one of the entities, the antigen-binding portion of the CAR recognizes and binds antigen in the extracellular domain of the CD22 protein. In one of the entities, the CD22 protein is expressed on a cancer cell. As used herein, the term "CD22" includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants, of full-length wild-type CD22.

The term "tumor antigen" encompasses "tumor-specific antigen" and "tumor-associated antigen". Tumor-specific antigens (TS A - Tumor-Specific Antigens) are usually present only on tumor cells and not on any other cells, while tumor-associated antigens (TAA Tumor-Associated Antigens) are present not only on some tumor cells but also on some normal cells. The tumor antigen of the present invention also refers to mutant forms of the protein that appear only in tumors of similar forms, while the unmutated form is observed in non-tumor tissues.

The term "extracellular antigen-binding domain" as used in the present invention refers to an oligo- or polypeptide capable of binding a specific antigen. Preferably, the domain can interact with a molecule on the cell surface. For example, the extracellular antigen-binding domain can be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In a particular case, said extracellular antigen-binding domain comprises a single-chain antibody fragment (scFv) comprising a light (Vl) and a heavy (Vh) variable fragment of a monoclonal antibody specific for a target antigen, which are connected by a flexible linker. The antigen binding domain of the CAR expressed on the cell surface of the engineered immune cells described in the present invention can be any domain that binds to a target antigen and that is derived from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof.

By "chimeric antigen receptor" or "CAR" is generally meant a synthetic receptor comprising a targeting moiety that binds to one or more signaling domains in a single fusion molecule. According to the present invention, the term "chimeric antigen receptor" encompasses both single-chain CARs and multichain CARs. Typically, the binding portion of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody, connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domains for first-generation CARs are derived from the cytoplasmic region of the CD8zeta or gamma chains of the Fc receptor. First-generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they cannot provide prolonged expansion and antitumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD 134) and 4-1BB (CD 137) have been added alone (second generation) or in combination (third generation) to improve survival and enhance proliferation of CAR-modified T cells. CARs are not necessarily single-chain polypeptides only, multi-chain CARs are also possible. According to the multi-chain CAR architecture, such as that described in WO 2014039523, the signaling domains and the co-stimulatory domains are located on different polypeptide chains. Such multichain CARs can be derived from FcsRI by replacing the high-affinity IgE-binding domain of the FcsRI alpha chain with an extracellular ligand-binding domain such as scFv, while the N- and/or C-terminal tails of the FcsRI beta and/or gamma chain are fused to signaling domains and costimulatory domains, respectively. The extracellular ligand-binding domain plays a role in redirecting T cell specificity to target cells, while the signaling domains activate the immune cell response.

By "immune cell" is meant a cell derived from a hematopoietic cell, functionally involved in the initiation and/or implementation of an innate and/or adoptive immune response, typically CD45, CD3 or CD4 positive cells. The immune cell described in the present invention can be a dendritic cell, a dendritic killer cell, a mast cell, a macrophage, a natural killer cell (NK cell), a cytokine-induced killer cell (CIK cell), a B cell or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes, gamma delta T cells, natural killer T cells (NKT cells).

By "allogeneic" we mean cells that are donor-derived or produced and/or differentiated from stem cells with a view to administration to patients with a different haplotype. Such immune cells are usually engineered to be less alloreactive and/or more resistant to their host patient. More specifically, the method for engineering allogeneic immune cells may include a step of reducing or inactivating TCR expression in T cells or in stem cells that are to be transformed into T cells. This may be achieved by various sequence-specific reagents, methods such as gene silencing or gene editing (nuclease, base editing, shRNA, RNAi, etc.).

The term "donor-derived" means that the T cells do not necessarily come directly from the donor as new cells, but may be derived from stem cells or cell lines derived from original donors who are not the treated patients (i.e. have different haplotypes).

By "primary cell" or "primary cells" is meant cells taken directly from living tissue (e.g., biopsy material) and intended to grow in vitro for a limited period of time, meaning that they can undergo a limited number of population doublings. Primary cells are contrasted with continuous tumorigenic or artificially immortalized cell lines. Non-limiting examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; HCO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; MOLT-4 cells.

Primary immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors, such as lymphocyte-infiltrated tumors. In some embodiments, the immune cells can be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In another embodiment, the cell is part of a mixed population of immune cells having different phenotypic characteristics, such as CD4, CD8, and CD56 positive cells. Primary immune cells are obtained from donors or patients using various methods known in the art, such as leukapheresis methods as reviewed by Schwartz J et al., J ClinApher., 2013, 28(3): 145-284.

Immune cells derived from stem cells are also considered as primary immune cells according to the present invention, in particular cells derived from induced pluripotent stem cells (iPS induced pluripotent stem cells) [Yamanaka, K. et al., Science, 2008, 322(5903): 949-953]. Lentiviral expression of reprogramming factors is used to induce multipotent cells from human peripheral blood cells [Staerk J. et al., Cell stem cell, 20107, (1): 20-24] [Loh Y.H. et al., Cell stem cell, 2010, 7 (1): 15-19].

Immune cells can be derived from human embryonic stem cells by methods well known in the art that do not involve the destruction of human embryos [Chung et al., Cell Stem Cell, 2008, 2(2): 113-117].

Genetic engineering refers to any method that introduces, modifies, and/or removes genetic material from a cell. Gene editing refers to genetic engineering that adds, deletes, or changes genetic material at specific locations (loci) in the genome, including point mutations. Gene editing typically involves sequence-specific reagents.

By "identity" is meant the identity of the sequences of two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that can be aligned for comparison. If a particular position in the sequence being compared is occupied by the same base, then the molecules at that position are identical. The degree of similarity or identity between nucleic acid or amino acid sequences depends on the number of identical or matching nucleotides at positions common to the nucleic acid sequences. Various algorithms and/or alignment programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, WI), and can be used, for example, with the default setting. For example, polypeptides that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the specific polypeptides described in the present invention, and preferably have substantially the same functions, as well as polynucleotides encoding such polypeptides. Unless otherwise specified, the present invention encompasses polypeptides and polynucleotides that have the same function and are at least 80%, typically at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% identical to the sequences described in the present invention.

The terms "patient", "subject" or "donor" as used herein include all members of the animal kingdom, including primates, including humans.

The present invention is based on the unexpected observation that a tumor cell expressing low levels of CD20 antigen and low levels of CD22 antigen, despite being unable to efficiently activate CAR-T cell targeting either CD20 or CD22, can activate CAR-T cells with two CARs expressing both a CD20-targeting CAR and a CD22-targeting CAR.

In these conditions, without relying on theory, the total number of CD20 antigen molecules and CD22 antigen molecules per tumor site represents the threshold above which dual-CAR CAR-T cells are activated, as opposed to single-CAR CAR-T cells.

This can be represented as follows:

X=density of CD20 antigen molecules on the tumor surface that are recognized by anti-CD20 CAR (CAR20);

Y=density of CD22 antigen molecules on the tumor surface that are recognized by anti-CD22 CAR (CAR22);

X+Y=Z=density of CD20 antigen molecules and CD22 antigen molecules on the tumor surface;

T=density of antigen molecules bound to a T cell that triggers the specified T cell activation=threshold required for T cell activation;

If Z>T, then T-cell activation occurs.

Thus, one of the advantages of the T cells of the present invention expressing both CAR20 and CAR22 (the dual receptor "CAR20x22 T cell" or "CAR22x20 T-ioieTKa" cells) is that they can be used in immunotherapy to target an expanded population of tumor cells having different levels of expression of the CD20 and/or CD22 antigens. Thus, not only tumor cells having a low level of expression of the CD20 antigen and a high level of expression of the CD22 antigen, as well as having a high level of expression of the CD20 antigen and a low level of expression of the CD22 antigen, will be targeted (and thus will be killed) by the dual receptor CAR22x20 T cells of the present invention, but, surprisingly, cells having low levels of expression of the CD20 and CD22 antigens will be targeted and thus will be killed.

Another advantage of the dual receptor CAR T cells of the present invention is their utility in immunotherapy for the treatment of a tumor that develops by expressing more or less CD20 and CD22 antigens, including during treatment.

Another advantage of the dual receptor CAR-T cells of the present invention is their utility in immunotherapy for the treatment of cancer characterized by low expression of CD20 and CD22 antigens.

Another advantage of the dual CAR T cells with two receptors of the present invention is related to the synergistic effect of the two CARs, possibly by favoring and enhancing the immune synapse between T cells and their target tumor cells, possibly providing a higher level of effector cytokine production.

In addition to the above-mentioned advantages, the dual receptor CAR T cells of the present invention are also useful for preventing CD20 and CD22-associated cancer recurrence and/or antigen escape.

What has been discovered and described in the present invention can likely be generalized such that CAR-T cells with two receptors targeting two different tumor-associated antigens may be useful in immunotherapy for targeting an expanded population of tumor cells having different levels of expression of tumor-associated antigens, such as those described in detail above.

Another unexpected effect of one of the objects disclosed in the present invention is related to the observation that an immune cell expressing CAR20 and CAR22 on its cell surface and having an exogenous nucleic acid integrated into the cell genome containing in the 5' to 3' direction:

(i) promoter,

(ii) a nucleic acid encoding said CAR20,

(iii) a nucleic acid encoding a self-cleaving peptide;

(iv) a nucleic acid encoding said CAR22,

wherein the same promoter controls the expression of the said CAR20 and CAR22;

demonstrates more significant tumor reduction in vivo compared to an immune cell expressing CAR20 and CAR22 on its cell surface and having an exogenous nucleic acid integrated into the cellular genome containing in the 5' to 3' direction:

(i) promoter,

(ii) a nucleic acid encoding said CAR22,

(iii) a nucleic acid encoding a self-cleaving peptide;

(iv) a nucleic acid encoding said CAR20. Immune cells expressing an anti-CP20 CAR and an anti-CP22 CAR The immune cells of the present invention are endowed with two synthetic chimeric antigen receptors (CARs) targeting the CD20 antigen and the CD22 antigen, respectively.

Anti-CP22 CAR (CAR22) and anti-CP20 CAR (CAR20)

The immune cells described in the present invention are endowed with two synthetic CARs, which gives them higher specificity with respect to cells, such as tumor cells expressing the CD20 and/or CD22 antigen, or to inflammatory cells expressing the CP20 and/or CP22 antigen.

The recombinant chimeric antigen receptor is typically encoded by an exogenous polynucleotide that is introduced into the cell using viral vectors according to one of the transduction steps also described herein. The recombinant receptor encoded by the exogenous polynucleotide can also be introduced into the cell as a plasmid or PCR product.

Typically, CAR polypeptides comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and/or a primary signaling domain, wherein said antigen-binding domain binds to an antigen associated with a disease state.

Although the anti-CP20 and anti-CP22 CARs described in the present invention are not limited to a particular CAR structure, a nucleic acid that can be used to genetically engineer immune cells typically encodes a CAR comprising: an extracellular antigen-binding domain that binds to an antigen associated with a disease state, a hinge, a transmembrane domain, and an intracellular domain comprising a stimulatory domain and/or a primary signaling domain. Typically, the extracellular antigen-binding domain is an scFv comprising a heavy variable chain (VH) and a light variable chain (VL) of an antibody that binds to a specific antigen (e.g., a tumor antigen) connected via a linker. The transmembrane domain can be, for example, the CB8α transmembrane domain or the 4-1BB transmembrane domain. The stimulatory domain can be, for example, the 4-1BB stimulatory domain. The primary signaling domain may be, for example, the CD3ξ signaling domain.

In one embodiment of the present invention, in order to avoid any recombination in a construct comprising polynucleotides encoding two CARs with identical domains, the nucleotide sequences used to encode the same amino acid sequences are present in the construct twice (e.g., the same transmembrane domain, the same stimulatory domain) are optimized using codons and taking into account the degeneracy of the code, so that the nucleotide sequences are different.

In one aspect of the present invention, the antigen-binding domain specific for CD20 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker, wherein said VH chain comprises the H-CDRs of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and said VL chain comprises the L-CDRs of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

For example, an antigen-binding domain specific for CD20 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker (forming the scFv of SEQ ID NO: 17), wherein said VH and VL chains comprise the H-CDRs of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49 and the L-CDRs of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

The anti-CD20 CAR described in the present invention may comprise:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15, comprising the H-CDRs of the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16, comprising the L-CDRs of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In one aspect of the present invention, a CAR targeting the CD20 antigen present on tumor cells is expressed by the genetically engineered immune cells described in the present invention, as described in Tables 3 and 4.

In one aspect of the present invention, the antigen-binding domain specific for CD22 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker, wherein said VH chain comprises the H-CDRs of SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and said VL chain comprises the L-CDRs of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46.

For example, an antigen-binding domain specific for CD22 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker (forming the scFv of SEQ ID NO: 13), wherein said VH and VL chains comprise the H-CDRs of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and the L-CDRs of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.

The anti-CD22 CAR of the present invention may comprise:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11, comprising the H-CDRs of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16, comprising the L-CDRs of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In one aspect of the present invention, a CAR targeting the CD22 antigen present on tumor cells is expressed by the genetically engineered immune cells described in the present invention as described in Tables 5 and 6 and below in the Examples section.

Immune cells expressing CAR20 and CAR22

The engineered immune cells described in the present invention are provided with two synthetic chimeric antigen receptors (CARs) targeting the CD20 antigen and the CD22 antigen, respectively, as described.

In a specific embodiment of the present invention, said immune cells do not express any other CAR targeting another antigen other than CD20 or CD22. More specifically, said immune cells do not express other CARs other than CAR22 and CAR20 described herein.

The immune cell may be, for example, a dendritic cell, a dendritic killer cell, a mast cell, a macrophage, an NK cell, a cytokine-induced killer cell, a B cell, or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes, gamma delta T cells, NKT cells, and TILL tumor infiltrating lymphocytes.

In a specific embodiment of the present invention, an immune cell engineered to express two CARs is selected from the group consisting of a T cell, an NK cell, and a macrophage.

In a specific embodiment of the present invention, the immune cell expressing two CARs is a T cell, such as a cytotoxic T cell.

In general, the immune cell in question consists of a population of cells, such as a population of immune cells, in particular a population of T cells, a population of NK cells, and/or a population of macrophages.

In a specific embodiment of the present invention, said immune cell is a T cell for use in immunotherapy as a finished drug.

In a specific embodiment of the present invention, said engineered immune cell is a T cell that is TCR negative (does not express TCR alpha on its cell surface).

In a specific embodiment of the present invention, said engineered T cell expresses a short hairpin RNA (shRNA) or small interfering RNA (siRNA) directed against a polynucleotide sequence encoding a TCR component.

In another specific embodiment of the present invention, said engineered T cell carries mutations in the TCR alpha alleles and/or the TCR beta alleles.

In another specific embodiment of the present invention, said engineered T cell may have at least one allele encoding TCR alpha, TCR beta and/or CD3 that is inactivated by mutation.

In another embodiment of the present invention, said engineered T cell has at least one allele selected from β2m, PD1, CTLA4, dCK, CD52 and/or GR that has been inactivated.

In another specific embodiment of the present invention, the engineered immune cell does not express the rituximab-specific mimotope of SEQ ID NO: 22.

Another specific embodiment of the present invention relates to genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises VH SEQ ID NO: 11 and VL SEQ ID NO: 12, and said CAR20 comprises VH SEQ ID NO: 15 and VL SEQ ID NO: 16.

Even more particularly, the present invention provides genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

A particular object of the present invention concerns genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, said CAR20 comprises the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16, and said engineered T cell has at least one allele encoding TCRalpha, TCRbeta and/or CD3 that is inactivated by mutation.

A particular aspect of the present invention relates to genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, said CAR20 comprises the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16, and said engineered T cell has at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation and has at least one selected allele of CD52 and p2m that have been inactivated, in particular wherein said engineered T cell has TCRalpha and/or TCRbeta and CD52 that have been inactivated by mutation.

Even more specifically, the present invention provides genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and said engineered T cell has at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation.

Even more particularly, the present invention provides genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and said engineered T cell has at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation, and has at least one allele selected from CD52 and β2m that has been inactivated, in particular wherein said engineered T cell has TCRalpha and/or TCRbeta and CD52 that have been inactivated by mutation.

The present invention also provides a genetically engineered immune cell expressing CAR20 and CAR22 on its cell surface as described above, wherein said CARs are encoded by an exogenous nucleic acid inserted into the genome of said immune cell, and said exogenous nucleic acid comprises in the 5' to 3' direction:

(i) a promoter, (for example, EF1 alpha promoter),

(ii) a nucleic acid encoding said CAR20,

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19),

(iv) a nucleic acid encoding said CAR22,

where the same promoter controls the expression of these CAR20 and CAR22.

In particular, a genetically engineered immune cell expressing CAR22 and CAR20 on its cell surface as described above is also provided, wherein said exogenous nucleic acid comprises in the 5' to 3' direction:

(i) a promoter (such as the EP1alpha promoter) that controls the expression of said CAR20;

(ii) a nucleic acid encoding said CAR20 comprising a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8alpha hinge of SEQ ID NO: 4, a CD8alpha transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9;

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19);

(iv) a nucleic acid encoding said CAR22 comprising a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CDS-alpha hinge of SEQ ID NO: 4, a CD8alpha transmembrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9;

where the same promoter controls the expression of these CAR20 and CAR22.

In another embodiment of the present invention, there is also provided a genetically engineered immune cell expressing CAR22 and CAR20 on its cell surface as described above, wherein said CARs are encoded by an exogenous nucleic acid included in the genome of said immune cell, and said exogenous nucleic acid comprises, in the 5' to 3' direction:

(i) a promoter (such as the EP1alpha promoter),

(ii) a nucleic acid encoding said CAR22,

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19),

(iv) a nucleic acid encoding said CAR20,

Moreover, the same promoter controls the expression of these CAR20 and CAR22.

In particular, a genetically engineered immune cell expressing CAR22 and CAR20 on its cell surface as described above is also provided, wherein said exogenous nucleic acid comprises in the 5' to 3' direction:

(i) a promoter (such as the EP1alpha promoter) that controls the expression of said CAR22,

(ii) a nucleic acid sequence encoding said CAR22 comprising the signal peptide of SEQ ID NO: 1, the scFv of SEQ ID NO: 13, the CD8alpha hinge of SEQ ID NO: 4, the CD8alpha transmembrane domain of SEQ ID NO: 6, the 4-IBB costimulatory domain of SEQ ID NO: 8 and the CD8zeta signaling domain of SEQ ID NO: 9),

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19),

(iv) a nucleic acid encoding said CAR20 comprising a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8alpha hinge of SEQ ID NO: 4, a CD8alpha transmembrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9.

Methods for Obtaining Genetically Engineered Immune Cells Immune cells that are to be genetically engineered to express CAR20 and CAR22 according to the description of the present invention can be obtained by introducing one or more exogenous polynucleotides encoding said CARs. Said polynucleotide can be introduced into a cell using viral vectors by the transduction method. Said polynucleotide can also be introduced into a cell as a plasmid or a PCR product.

Stable expression of CARs, in particular CAR20 and CAR22, described herein, in the immune cells described above can be achieved using, for example, viral vectors (e.g., lentiviral vectors, retroviral vectors, adeno-associated virus (AAV) vectors) or by integrating transposons/transposase systems or plasmids or PCR products. Other approaches include direct electroporation of mRNA.

To simultaneously deliver both CARs into a cell, the polynucleotide(s) encoding the anti-CD22 CAR (CAR22) and anti-CD20 CAR (CAR20) described in the present invention may have different structures, such as:

(a) a polycistronic structure in which both two transcriptional units are controlled by a unique promoter, have the same direction of transcription, and are separated by a "self-cleaving" peptide such as peptide 2A (e.g., P2A, T2A, E2A, F2A),

(b) a bidirectional arrangement in which each of the 2 transcription units is controlled by an independent promoter in a head-to-head configuration and which are transcribed in opposite directions, and

(c) monocistronic arrangement with both scFV transcripts separated by a genomic spacer.

As used herein, the term "polycistronic" mRNA refers to a single messenger RNA that contains two or more coding sequences (i.e., cistrons) and encodes more than one protein. Polycistronic mRNA may contain any element known in the art that provides for the translation of two or more genes from the same mRNA molecule, including, but not limited to, a self-cleaving peptide such as a P2A element, a T2A element, an E2A element, and an F2A element, or an IRES element.

The self-cleaving peptide to be included in the polynucleotides of the present invention may be selected from peptide 2A, 2A-like peptide, P2A peptide, E2A peptide, F2A peptide, T2A peptide, in particular peptide 2A, more particularly peptide P2A of SEQ ID NO: 19, peptide T2A of SEQ ID NO: 38, peptide E2A of SEQ ID NO: 39 or peptide F2A of SEQ ID NO: 40, even more particularly peptide P2A of SEQ ID NO: 19.

The abbreviation IRES (Internal ribosome entry site) means "internal ribosome entry site", and any IRES that allows transcription and then translation of the coding sequence inserted into the gene can be used according to the present invention. For example, an IRES included in the polynucleotides described in the present invention can have SEQ ID NO: 37.

As described in the present invention, CAR20 and CAR22 may be encoded by two nucleic acids, where:

1) one nucleic acid encodes CAR20, which contains:

1) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

2) the other nucleic acid encodes CAR22 comprising: i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 12 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In one embodiment, CAR22 and CAR20 are encoded by two independent nucleic acids a) and b), where:

(1) Nucleic acid a) encodes CAR22, which contains:

i) at least one extracellular domain comprising:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

(2) nucleic acid b) encodes CAR20, which contains:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In a specific embodiment of the present invention, there is provided an isolated polynucleotide comprising:

a) a nucleic acid encoding CAR22, comprising: i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain, and

b) nucleic acid encoding CAR20, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

In another embodiment, nucleic acids a) and b) are located in a single nucleic acid molecule, and the isolated polynucleotide contains a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A, F2A) located between nucleic acids a) and b).

Thus, the main object of the present invention is a polynucleotide comprising in the 5' to 3' direction:

(i) a promoter (such as the EP1alpha promoter)

(ii) a nucleic acid encoding CAR20 as described in the present invention,

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19);

(iv) a nucleic acid encoding CAR22 as described in the present invention,

where the same promoter controls the expression of these CAR20 and CAR22.

The present description also provides a polynucleotide comprising in the 5' to 3' direction:

(i) a promoter (such as the EP1alpha promoter)

(ii) a nucleic acid encoding CAR22 as described in the present invention,

(iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19);

(iv) a nucleic acid encoding CAR20 as described in the present invention,

where the same promoter controls the expression of these CAR20 and CAR22.

In one embodiment of the present invention, the isolated polynucleotide described in the present invention does not comprise a nucleic acid encoding a CAR other than CAR22 and CAR20.

In one embodiment of the present invention, the isolated polynucleotide described in the present invention does not comprise a nucleic acid encoding the rituximab-specific mimotope of SEQ ID NO: 22.

In one embodiment of the present invention, an isolated polynucleotide encoding CAR20 and CAR22 as described herein comprises:

a) a promoter (such as the EE1alpha promoter) that controls the expression of said CAR20, followed by a nucleic acid encoding said CAR20, wherein said CAR20 comprises:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; and

b) nucleic acid encoding CAR22, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 12 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain;

wherein said nucleic acids a) and b) are located in the same nucleic acid molecule, and wherein a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A or F2A) is located between said nucleic acids a) and b).

In another embodiment of the present invention, an isolated polynucleotide encoding CAR20 and CAR22 of the present invention comprises:

(a) a promoter (such as the EE1alpha promoter) that controls the expression of said CAR22, followed by a nucleic acid encoding said CAR22, wherein said CAR22 consists of:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 11 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 12 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

b) nucleic acid encoding CAR20, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15 and comprises the H-CDRs of the amino acid sequences of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 16 and comprises the L-CDRs of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: ID NO: 51 and SEQ ID NO: 52, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain;

wherein said nucleic acids a) and b) are located in the same nucleic acid molecule, and wherein a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A or F2A) is located between said nucleic acids a) and b).

In another embodiment of the present invention, an isolated polynucleotide encoding CAR20 and CAR22 as described herein comprises:

a) a nucleic acid encoding CAR22, which comprises a promoter (such as the EF1 alpha promoter) controlling the expression of said CAR22, consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8alpha hinge of SEQ ID NO: 4, a CDSalpha transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9;

b) a nucleic acid encoding CAR20, consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CDS-alpha hinge of SEQ ID NO: 4, a CDS-alpha trans membrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, and a CD3 zeta signaling domain of SEQ ID NO: 9; and a CD8 zeta signaling domain of SEQ ID NO: 9; and

c) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) placed between nucleic acid a) and nucleic acid b), allowing for the simultaneous expression of said CAR20 and CAR22.

In another embodiment of the present invention, an isolated polynucleotide encoding CAR20 and CAR22 as described herein comprises:

a) a nucleic acid encoding CAR20, which comprises a promoter (such as the EF1 alpha promoter) controlling the expression of said CAR20, consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8alpha hinge of SEQ ID NO: 4, a CD8alpha transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9;

b) a nucleic acid encoding CAR22, consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CDS-alpha hinge of SEQ ID NO: 4, a CD8-alpha trans membrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9; and

c) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) placed between nucleic acid a) and nucleic acid b), allowing for the simultaneous expression of said CAR22 and CAR20.

In another embodiment, the present invention provides an isolated polynucleotide encoding CAR20 and CAR22 comprising the nucleic acid sequence of SEQ ID NO: 31 (CAR22xCAR20 construct).

In another specific embodiment, the present invention provides an isolated polynucleotide encoding CAR20 and CAR22 comprising the nucleic acid sequence of SEQ ID NO: 32 (CAR20xCAR22 construct).

Also provided in the present invention is a vector comprising any of the isolated polynucleotides of the present invention.

Also provided in the present invention is a host cell comprising a vector of the present invention.

The immune cell population to be engineered is typically isolated from the blood of a patient or healthy donor by apheresis and further engineered to express the chimeric antigen receptor on their surface. Alternatively, the immune cell population to be engineered may be derived from cord blood cells or from stem cells that are further engineered to express the chimeric antigen receptor on their surface.

Said CAR-expressing immune cells may be derived from immune cells of the patient or a compatible donor that have been engineered to express a specific CAR on their surface.

These CAR-expressing immune cells may also be derived from stem cells such as iPS cells derived from the patient or a compatible donor, or from tumor infiltrating lymphocytes (TILL).

In other aspects, the CAR-expressing immune cells are so-called “off-the-shelf” immune cell formulations, in which immune cells not specifically from the patient to be treated have been engineered to express CAR and become suitable for allogeneic therapeutic treatment.

The term "allogeneic" means that the cells are derived from a donor or are produced and/or differentiated from stem cells with the intention of being administered to patients with a different haplotype.

Such immune cells are usually engineered to be less alloreactive and/or more resistant to their host patient. More specifically, the method for engineering allogeneic immune cells may involve a step of reducing or inactivating TCR expression in T cells or in stem cells that are to be transformed into T cells. This can be achieved by various sequence-specific reagents, such as gene silencing or gene editing (nuclease, base editing, RNAi…).

Reliable gene editing protocols and strategies for the generation of allogeneic therapeutic grade T cells from PBMCs have been previously reported, especially with the use of highly safe and specific endonuclease reagents in the form of TALE nucleases (TALEN®). The generation of so-called “universal 1 cells”, which are [TCR] ^negative T cells, from donors has been achieved and successfully administered to patients with reduced graft versus host disease (GVhD) (Poirot et al., Cancer. Res. 2015, 75 (18): 3853-3864; Qasim et al., Science Translational 2017, 9(374)). Meanwhile, inactivation of TCR or β2m components in primary T cells can be combined with inactivation of additional genes encoding checkpoint inhibitor proteins, as described, for example, in WO 2014184744.

In other cases, the engineered immune cell may be further modified to confer resistance to at least one immunosuppressive drug, such as by inactivating CD52, which is the target of an anti-CD52 antibody (e.g., alemtuzumab), as described, for example, in WO2013176915.

In other cases, the engineered immune cell may be further modified to confer resistance to a chemotherapeutic agent, in particular a purine analogue drug, for example by inactivating DCK as described in WO 201575195.

In other cases, the engineered immune cell may be further modified to improve its persistence or lifespan in the patient, in particular by inactivating the gene encoding the MHC-I component(s), such as HLA or β2m, as described in WO 2015136001 or in the publication by Liu et al. (Cell Res 2017, 27:154-157).

In other cases, the engineered immune cell is mutated to improve its CAR-dependent immune activation, in particular to reduce or suppress the expression of immune checkpoint proteins and/or their receptors, such as PD1 or CTLA4, as described in WO 2014184744.

Pharmaceutical formulations

One aspect of the present invention relates to a pharmaceutical composition comprising genetically engineered immune cells expressing on their cell surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) according to the present invention, and a pharmaceutically acceptable excipient.

A pharmaceutical composition has also been developed, comprising a population of immune cells, including genetically engineered immune cells expressing on their cell surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20), described in the present invention, and a pharmaceutically acceptable excipient.

A particular object of the present invention relates to a pharmaceutical composition comprising genetically engineered T cells of the present invention and a pharmaceutically acceptable excipient.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells of the present invention and a pharmaceutically acceptable excipient.

Another particular subject of the present invention relates to a pharmaceutical composition comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises VH SEQ ID NO: 11 and VL SEQ ID NO: 12, and said CAR20 comprises VH SEQ ID NO: 15 and VL SEQ ID NO: 16.

Even more particularly, a pharmaceutical composition is provided comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises VH SEQ ID NO: 11 and VL SEQ ID NO: 12, and said CAR20 comprises VH SEQ ID NO: 15 and VL SEQ ID NO: 16.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and said CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

Another particular aspect of the present invention relates to a pharmaceutical composition comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises VH SEQ ID NO: 11 and VL SEQ ID NO: 12, and said CAR20 comprises VH SEQ ID NO: 15 and VL SEQ ID NO: 16, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation, and/or wherein said engineered T cells have at least one inactivated allele selected from (32m and CD52.

Even more particularly, there is provided a pharmaceutical composition comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that is inactivated by mutation.

Even more particularly, there is provided a pharmaceutical composition comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation, and/or wherein said engineered T cells have at least one allele selected from β2m and CD52 that has been inactivated.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises VH SEQ ID NO: 11 and VL SEQ ID NO: 12, said CAR20 comprises VH SEQ ID NO: 15 and VL SEQ ID NO: 16, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation, and/or wherein said engineered T cells have at least one allele selected from β2m and CD52 that has been inactivated.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation.

Another particular object of the present invention relates to a pharmaceutical composition comprising a population of T cells comprising genetically engineered T cells expressing CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, wherein said CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and wherein said T cells have at least one allele encoding TCRalpha, TCRbeta and/or CD3 that has been inactivated by mutation, and/or wherein said engineered T cells have at least one allele selected from (32m and CD52 that has been inactivated.

The present invention also provides genetically engineered immune cells expressing CAR20 and CAR22 as described herein for use as a medicament.

The present invention also provides a population of immune cells comprising genetically engineered immune cells expressing CAR20 and CAR22 as described herein for use as a medicament.

Treatment methods

Another object relates to methods of treating cancer and/or an inflammatory disease comprising administering to a patient in need thereof genetically engineered immune cells expressing CAR20 and CAR22 as described herein.

A similar object concerns genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention for use in a method of treating cancer and/or an inflammatory disease.

A similar subject matter relates to a population of immune cells comprising genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention for use in a method of treating cancer and/or an inflammatory disease.

Similar objects relate to the use of genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention, or a population of immune cells comprising genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention, for the preparation of a medicament.

A similar object concerns the use of genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention, or a population of immune cells comprising genetically engineered immune cells expressing CAR20 and CAR22 as described in the present invention, for the preparation of a medicament for the treatment of cancer and/or an inflammatory disease.

The treatment may be for the treatment of cancer, including hematological cancer, such as a hematological cancer selected from lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) (also known as acute lymphoblastic leukemia), acute lymphocytic cancer, acute myeloid leukemia (AML), in particular CD22- and/or CD20-associated hematological cancer, more particularly refractory to relapse CD22- and/or CD20-associated hematological cancer, even more particularly an aggressive form of said CD22- and/or CD20-associated hematological cancer.

Treatment may involve preventing or attenuating CD20 and/or CD22-associated inflammatory disease.

In this specific setting, the treatment is intended to treat patients with relapse-refractory NHL.

In another specific target, the treatment is intended to treat patients with cancer who have low expression of the CD20 and CD22 antigens.

In this specific target, the treatment is intended to treat patients who have previously been treated with rituximab (an anti-CD20 antibody that is the standard of care for NHL).

“Non-Hodgkin lymphoma (NHL)” is a term for a group of diverse blood cancers that have one thing in common: they all arise from lymphocytes. The World Health Organization (WHO) has identified more than 60 specific subtypes of NHL, which have been given names as “diagnostic designations.”

In particular, the term "non-Hodgkin lymphoma (NHL)" includes the following diagnostic designations for non-Hodgkin lymphoma (NHL):

1. Mature B-cell lymphomas (about 85-90% of NHL cases):

- Aggressive: diffuse large B-cell lymphoma (DLBCL) (31%), mantle cell lymphoma (MCL) (may present as aggressive or indolent) (6%), lymphoblastic lymphoma (2%), Burkitt lymphoma (BL) (2%). %), primary mediastinal (thymic) large B-cell lymphoma (PMLBCL) (2%), transformed follicular and transformed mucosa-associated lymphoid tissue (MALT), high-grade B-cell lymphoma with double or triple involvement (HBL), primary cutaneous DLBCL (leg type), primary DLBCL of the central nervous system, primary lymphoma of the central nervous system (CNS), lymphoma associated with acquired immunodeficiency syndrome (AIDS)

Indolent: follicular lymphoma (FL) (22%), marginal zone lymphoma (MZL) (8%), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (6%), gastric mucosa-associated lymphoid tissue lymphoma (GMLAT) (5%), lymphoplasmacytic lymphoma (1%), Waldenstrom's macroglobulinemia (WM), nodular marginal zone lymphoma (NML) (1%), splenic marginal zone lymphoma (SMZL),

2. Mature T-cell lymphomas and natural killer (NK) cell lymphomas (about 10-15% of NHL cases)

- Aggressive: peripheral T-cell lymphoma (PTCL), not otherwise specified (6%), systemic anaplastic large cell lymphoma (ALCL) (2%), lymphoblastic lymphoma (2%), hepatosplenic gamma/delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma (SPLTCL), intestinal T-cell lymphoma of enteropathic type, primary cutaneous anaplastic large cell lymphoma

- Indolent: cutaneous T-cell lymphoma (CTCL) (4%), mycosis fungoides (MF), Sezary syndrome (SS), angioimmunoblastic T-cell lymphoma (AITL), adult T-cell leukemia/lymphoma, extranodal NK/T-cell lymphoma (3NK/TKJI), nasal type.

The foregoing description of the present invention provides a method and process for its implementation and use in such a way that any person skilled in the art can make and use it, and this possibility is provided, in particular, in the essence of the appended claims, which form part of the original description.

Where a numerical limit or range is stated in this specification, the end points are included. In addition, all values and subranges within the numerical limit or range are expressly included as if they were expressly stated.

Having generally described the present invention, a further understanding thereof may be obtained by reference to certain specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the claimed invention.

Specific embodiments of the present invention

1. A genetically engineered T cell expressing on its cell surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20),

a) where the said CAR22 contains:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, and optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; and

b) where the said CAR20 contains:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain; and

c) wherein said engineered T cell is TCR-negative.

2. A genetically engineered T cell according to an embodiment of the present invention according to claim 1, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and said CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

3. A genetically engineered T cell according to any one of the embodiments of the present invention according to claims 1 and 2, wherein none of the extracellular domains of said CAR20 and CAR22 comprises the rituximab-specific mimotope of SEQ ID NO: 22.

4. A genetically engineered T cell according to any one of the embodiments of the present invention according to claims 1-3, wherein said engineered T cell expresses a short hairpin RNA (shRNA) or a small interfering RNA (siRNA) directed against a polynucleotide sequence encoding a TCR component.

5. A genetically engineered T cell according to any one of the embodiments of the present invention according to claims 1-3, wherein in said engineered T cell the TCRalpha and/or TCRbeta allele is mutated.

6. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-3 and 5, wherein in said engineered T cell at least one allele encoding TCR alpha, TCR beta and/or CD3 is inactivated by mutation.

7. A genetically engineered T cell according to any one of the embodiments of the present invention according to claims 1-6, wherein said engineered T cell has an inactivated CD52 allele.

8. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-7, wherein said engineered T cell has an inactivated β2m allele.

9. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-8, wherein said engineered T cell has an inactivated PD1 allele.

10. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs. 1-9, wherein said engineered T cell has an inactivated CTLA4 allele.

11. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs. 1-10, wherein said engineered T cell has an inactivated dCK allele.

12. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-11, wherein said engineered T cell has an inactivated GR allele.

13. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-12, wherein said engineered immune cell is a cytotoxic T cell.

14. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-13, wherein said engineered T cell is part of a population of T cells.

15. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-14, wherein said engineered T cell is a primary cell.

16. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-15, wherein said engineered T cell is a mammalian cell, preferably a human cell.

17. A genetically engineered T cell according to any one of the embodiments of the present invention according to paragraphs 1-16, wherein said engineered T cell does not express another CAR besides said CAR22 and CAR20.

18. A population of T cells comprising engineered T cells according to any of the embodiments of the present invention according to paragraphs 1-7.

19. A pharmaceutical composition comprising engineered T cells according to any one of the embodiments of the present invention according to paragraphs 1-17 and a pharmaceutically acceptable excipient.

20. A pharmaceutical composition comprising a population of T cells according to the embodiment of the present invention according to claim 18 and a pharmaceutically acceptable excipient.

21. An isolated polynucleotide containing:

a) nucleic acid encoding CAR22, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and

CD8zeta signaling domain; and

b) nucleic acid encoding CAR20, containing:

i) at least one extracellular domain comprising:

- an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) SEQ ID NO: 15 and a variable light chain (VL)

SEQ ID NO: 16, optionally a leader sequence,

- hinge domain of CD8alpha,

ii) the transmembrane domain of CD8alpha, and

iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD8zeta signaling domain.

22. A polynucleotide according to an embodiment of the present invention according to claim 21, wherein nucleic acids a) and b) are in a single nucleic acid molecule, and the nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A or F2A) is located between nucleic acids a) and b).

23. A polynucleotide according to the embodiments of the present invention according to claims 21 or 22, wherein said individual polynucleotide does not contain a nucleic acid encoding a CAR other than said CAR22 and CAR20.

24. A polynucleotide according to embodiments of the present invention according to claims 21-23, wherein said single polynucleotide does not contain a nucleic acid encoding a rituximab-specific mimotope of SEQ ID NO: 22.

25. A polynucleotide according to any one of the embodiments of the present invention according to paragraphs 21-24, wherein the nucleic acid a) comprises a promoter (such as the EF1 alpha promoter) that controls the expression of said CAR22, consisting of the signal peptide of SEQ ID NO: 1, the scFv of SEQ ID NO: 13, the CD8alpha hinge of SEQ ID NO: 4, the CD8alpha transmembrane domain of SEQ ID NO: 6, the 4-1BB costimulatory domain of SEQ ID NO: 8 and the CD8zeta signaling domain of SEQ ID NO: 9;

wherein nucleic acid b) encodes said CAR20 consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8alpha hinge of SEQ ID NO: 4, a CDSalpha transmembrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, and a CD8zeta signaling domain of SEQ ID NO: 9; 20 wherein a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) is located between nucleic acid a) and nucleic acid b), allowing for the simultaneous expression of said CAR20 and CAR22.

26. A polynucleotide according to any one of the embodiments of the present invention according to paragraphs 21-24, wherein nucleic acid b) comprises a promoter (such as the EF1 alpha promoter) that controls the expression of said CAR20,

consisting of a signal peptide SEQ ID NO: 1, scFv SEQ ID NO: 17, CD8alpha hinge SEQ ID NO: 4, CD8alpha transmembrane domain SEQ ID NO: 6, 4-1BB costimulatory domain SEQ ID NO: 8 and CD8zeta signaling domain SEQ ID NO: 9;

wherein nucleic acid a) encodes said CAR22, consisting of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8alpha hinge of SEQ ID NO: 4, a CDSalpha transmembrane domain of SEQ ID NO: 6, a 4-IBB costimulatory domain of SEQ ID NO: 8, a CD8zeta signaling domain of SEQ ID NO: 9; wherein a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) is located between nucleic acid a) and nucleic acid b), allowing for the simultaneous expression of said CAR22 and CAR20.

27. An isolated polynucleotide according to an embodiment of the present invention according to claim 25, comprising the nucleic acid sequence of SEQ ID NO: 31 (the CAR22×CAR20 construct).

28. An isolated polynucleotide according to an embodiment of the present invention according to claim 26, comprising the nucleic acid sequence of SEQ ID NO: 32 (the CAR20×CAR22 construct).

29. A vector containing an isolated polynucleotide according to any of the embodiments of the present invention according to paragraphs 21-28.

30. A host cell comprising a vector according to an embodiment of the present invention according to claim 29.

31. A method for producing engineered T cells according to any one of the embodiments of the present invention according to paragraphs 1-17, comprising introducing into an immune cell a polynucleotide according to any one of the embodiments of the present invention according to paragraphs 21-28 or a vector according to the embodiment of the present invention according to paragraph 29.

32. Engineered T cells according to any of the embodiments of the present invention according to paragraphs 1-17 for use as a medicine.

33. Engineered T cells according to any one of the embodiments of the present invention according to paragraphs 1-17 for use in the treatment of cancer associated with the expression of CD20 and/or CD22.

34. Engineered T cells according to any one of the embodiments of the present invention according to claims 1-17 for use in the treatment of hematological cancer, in particular CD22- and/or CD20-associated hematological cancer, more particularly relapsed refractory CD22- and/or CD20-associated hematological cancer, even more particularly an aggressive form of said CD22- and/or CD20-associated hematological cancer.

35. Engineered T cells for use in accordance with the embodiments of the present invention according to paragraph 33 or 34, wherein said cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphocytic cancer, acute myeloid leukemia (AML).

36. Engineered T cells for use in accordance with the embodiment of the present invention according to claim 35, wherein said cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia.

37. Engineered T cells for use in accordance with any of the embodiments of the present invention according to paragraphs 33-36, wherein said cancer is associated with low expression of CD20 and/or CD22.

38. Engineered T cells for use in accordance with any of the 10 embodiments of the present invention according to paragraphs 33-37, wherein said cancer is relapsed non-Hodgkin's lymphoma.

39. A method for treating a patient suffering from cancer associated with the expression of CD20 and/or CD22, comprising administering to said patient an effective amount of engineered T cells according to any one of the embodiments of the present invention according to paragraphs 1-17 or a population of T cells according to paragraph 18.

40. The method of treatment according to the embodiment of the present invention according to claim 39, wherein said cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphocytic cancer, acute myeloid leukemia (AML).

41. A method of treatment according to an embodiment of the present invention according to claim 39 or 40, wherein said cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia.

42. A method of treatment according to any one of the embodiments of the present invention according to paragraphs 39-41, wherein said cancer is associated with low expression of CD20 and/or CD22.

43. A method of treatment according to any one of the embodiments of the present invention according to paragraphs 39-42, wherein said cancer is recurrent non-Hodgkin's lymphoma.

Examples

The examples provided herein show how to generate dual CAR-T cells targeting CD20 and CD22 antigens (“CD20×CD22” or “CD20×CD22” CAR-T cells) and demonstrate their ability to lyse tumor cells with low or no expression of CD22 and/or CD20 receptors.

Example 1. Generation of Dual CD20 and CD22 CAR Constructs and Their Control Two orientations of the dual CAR are designed and constructed as a recombinant lentiviral vector to compare expression, activity, and potency. The first dual construct (CD22xCD20) contains the EF1alpha promoter, which drives the expression of the first CD22 CAR, consisting of a signal peptide (SEQ ID NO: 1), scFv SEQ ID NO: 13, CD8alpha hinge (SEQ ID NO: 4) and TM domain (SEQ ID NO: 6), 4-IBB costimulatory domain (SEQ ID NO: 8) and CD8zeta signaling domain SEQ ID NO: 9. This first CAR is followed by the self-cleaving peptide P2A (SEQ ID NO: 19), which drives the expression of the second CD20 CAR, consisting of a signal peptide (SEQ ID NO: 1), anti-CD20 scFv SEQ ID NO: 17, CD8alpha hinge (SEQ ID NO: 4) and TM domain (SEQ ID NO: 6), 4-IBB costimulatory domain (SEQ ID NO: 8) and the CD8zeta signaling domain SEQ ID NO: 9. To avoid any recombination in the rLV construct, the nucleotide sequences used to encode the same amino acid sequences present twice in the construct are optimized for codon usage and code degeneracy in such a way as to dilute the nucleotide sequences. The second dual construct (CD20xCD22) contains exactly the same sequences (nucleotides and amino acids), but the CD20 CAR is located immediately downstream of the EF1 alpha promoter.

For comparison, CD22 or CD20 CARs were inserted into a recombinant lentiviral vector. These constructs contain the EPalpha promoter driving the expression of a single CAR targeting CD22 (SEQ ID NO: 14) or CD20 (SEQ ID NO: 18). Example 2. Generation of Dual CD20xCD22 or CD22xCD20 CAR T Cells Cryopreserved PBMCs from at least 3 different donors were used. PBMCs were thawed at 37°C, washed, and resuspended in OpTmizer medium supplemented with recombinant human interleukin-2 (rhIL-2, 350 IU/ml), human AB serum (5%), and incubated overnight at 37°C in a 5% _CO2 incubator. The cells were then activated with anti-CD3/CD28 coated beads in OpTmizer medium supplemented with human AB serum (5%) (and additional 5% CTS™ Immune Cell SR during the growth phase) and recombinant human interleukin-2 (rhIL-2, 350 IU/ml) in a _CO2 incubator for 3 days. The amplified T cells were then transduced with lentiviral particles expressing CD20xCD22 or CD22xCD20 CAR (SEQ ID NO: 31 and SEQ ID NO: 32, respectively) at an MOI of 15 in the presence of Lentiboost transduction enhancer (Mayflower bioscience SB-P-LV-101-12). Amplified T cells were also transduced with lentiviral particles expressing only the CD20 CAR or the CD22 CAR (SEQ ID NO: 14 and SEQ ID NO: 18) at an MOI of 5. Two days after transduction, the cells were electroporated with four mRNAs, two encoding the TRAC T01 TALEN arms (SEQ ID NO: 23 and SEQ ID NO: 24) and two encoding the CD52 T01 TALEN arms (SEQ ID NO: 25 and SEQ ID NO: 26), using the AgilePulse Max system. Cells were resuspended in culture medium, incubated for 16 to 18 h at 30°C, and expanded at 37°C after addition of fresh culture medium, adjusting the cell concentration periodically. On the last day of culture (18 days after thawing), T cells are used in various assays or frozen in freezing medium (90% FBS, 10% DMSO). Cells are stored frozen at -150°C until use.

Example 3. Detection of dual CD20 and CD22 CAR-T cells

To detect CD20 CAR, recombinant CD20 protein fused to a His-tag (Acgo, catalog number #CD0-H52H3, SEQ ID NO: 20) is used in combination with APC-tagged anti-His-antibody (BioLegend, catalog number #362605). To detect CD22 CAR, CD22-Fc protein (SEQ ID NO: 21) is used in combination with the Cy3 subclass 1 anti-Fcgamma tag (Jackson ImmunoResearch, catalog number #115-165-205).

Various CAR-T cells (non-transduced, CD20×CD22 CAR, CD22×CD20 CAR or CD22 CAR) obtained in Example 1 were incubated with 100 ng of CD22-Fc protein and 200 ng of CD20-His protein, washed and further incubated with anti-Fc gamma (50 ng) or anti-His (50 ng), then fixed in 2% PFA. The cells were then analyzed by flow cytometry.

The results in Fig. 1 show that transduction of CD22 CAR alone resulted in 40% CD22 CAR positive T cells, whereas transduction of CD22×CD20 or CD20×CD22 CAR resulted in 39% and 25% double positive CAR T cells, respectively. These results are reproducible using three different donors. Interestingly, CD22×CD20 resulted in increased mean fluorescent intensity (MFI) of CD22 CAR staining, whereas CD20×CD22 resulted in higher MFI of CD20 CAR staining.

Example 4: Cell lines used to test dual chimeric antigen receptor T cells (dual CD20 and CD22 CAR T cells)

The Raji cell line expresses high levels of CD20 and CD22. This cell line was modified to express the luciferase gene and is used as a positive control, i.e., for target cells expressing both CD20 and CD22. In addition, Raji cell lines are treated with CD22 and/or CD20 TALENs. Briefly, Raji cells were electroporated with mRNA encoding the CD22 TALEN arms (SEQ ID NO: 27 and SEQ ID NO: 28) and mRNA encoding the CD20 TALENs (SEQ ID NO: 29 and SEQ ID NO: 30) using the AgilePulse Max system. Cells were incubated at 30°C for 16-18 h in RPMI1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptavidin (culture medium) and grown in fresh culture medium at 37°C in a CO2 incubator until sorting. Different cell populations were purified using anti-biotin microbeads and either anti-CD20-biotin or anti-CD22-biotin or both (Biolegend) to obtain different cell populations expressing i) CD20 but not CD22, ii) CD22 but not CD20, or iii) neither CD20 nor CD22. Fig. 2 illustrates the phenotype of these selected populations.

Example 5. Cytotoxicity of Dual CD20 and CD22 CAR-T Cells CD20×CD22, CD22×CD20, and CD22 CAR-T cells obtained in Example 1 and obtained from three different donors were tested for cytotoxicity against different Raji cells obtained in Example 4.

T cells were thawed and viable cells were counted using a NucleoCounter® (Chemometec, model NC-250). Different CAR-T cells were co-cultured for 4 to 16 h (at 37°C in a 5% CO ₂ incubator) with different Raji cells (CD20+CD22+, CD20+CD22-, CD22-CD20+, and CD20-CD22-) expressing luciferase in 96-well plates at different effector/target ratios. At the end of the incubation period, released luciferase was measured using the One-Glo® kit (Promega, catalog #E6110) according to the supplier's protocol. In Fig. 3 shows that for all donors used, CD22 CAR-T cells can efficiently lyse CD20+CD22+ and CD20-CD22+ Raji clones, whereas CD22×CD20 or CD20×CD22 CAR-T cells provide the same level of lysis as CD22 CAR but can also lyse CD20 to the same level of+CD22- Raji clones.

The killing ability of CD20×CD22, CD22×CD20, and CD22 CAR-T cells obtained in Example 1 was also tested in a rechallenge or serial killing assay. Different CAR-T cells were thawed, counted, and incubated with different Raji cells at a 1:1 ratio of CAR-positive T cells to Raji cells (CD20+CD22+, CD20-CD22+, CD20+CD22-). After 3 days of incubation, half of the wells were used to measure luciferase release using the One-Glo® kit (Promega, catalog #E6110), and the other half were transferred to new plates containing Raji cells for additional incubation. Luciferase measurements were repeated after 7 days, 10 days, 14 days, and 17 days.

Figure 4 shows that CD22×CD20 or CD20×CD22 CAR-T cells are able to efficiently kill all Raji cells tested, whereas CD22 CAR-T cells are able to efficiently kill only CD20+CD22+ and CD20−CD22+ Raji cells. Surprisingly, CD22 CAR-T cells were shown to have low activity against CD20+CD22− Raji cells, confirming that CD22 expression is not completely abolished in this clone. This also suggests that dual CARs can efficiently kill target tumor cells with low expression of both target antigens.

Example 6. Tumor load control of a disseminated B-cell lymphoma model in vivo

Daudi cells expressing CD20 and CD22 were modified to express luciferase and GFP. Daudi cells were injected intravenously into NSG immunodeficient mice (NOD.Cg-Prkdcscid I12rgtml Wjl/SzJ, Jackson laboratories). Seven days after tumor implantation, individual mice were injected intravenously with CD20xCD22 (1 and 3 million), CD22xCD20 (1 and 3 million), and CD22 (10 million) CAR-T cells. The bioluminescence (BL) signal after D-luciferin injection was monitored biweekly until day 60 after CAR-T cell injection; the results are shown in Fig. 5. For all treatment conditions with the tested CART cells, the DLI signal was reduced compared with the vehicle control or untransduced T cells. Surprisingly, at a low dose of 1 million CAR+ T cells, CD20×CD22 CAR T cells exhibited potent killer activity that was greater than that of CD22×CD20 CAR-T cells.

Example 7. Treatment efficacy in an in vivo model of disseminated B-cell lymphoma

Animals injected with Daudi cells and subsequently injected intravenously with the CAR-T cells from Example 6 CD20xCD22 (1 million), CD22xCD20 (1 million), and CD22 (10 million) were monitored for survival for 60 days. As shown in Fig. 6, animals receiving any of the CAR-T cells tested survived longer than animals receiving either vehicle or untransduced T cells. Additionally, and unexpectedly, animals receiving CD20xCD22 CAR-T cells survived longer than animals receiving CD22xCD20 CAR-T cells.

Example 8. Treatment efficacy in an in vivo model of disseminated B-cell lymphoma

The Raji cell lines described in Example 2 were injected subcutaneously into NSG immunodeficient mice (NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ, Jackson Laboratories). All three cell lines were injected simultaneously into each animal, specifically, wild-type (WT) Raji cells were injected into one flank, CD22− Raji cells were injected into the other flank, and CD20− cells were also injected into the flank but at a different location. One week after tumor injection, CD20×CD22 (3 and 8 million), CD22×CD20 (3 and 8 million), and CD22 (8 million) CAR T cells were injected intravenously, and the survival of the animals was observed. As shown in Fig. 7, animals treated with CD22 CAR T cells rapidly succumbed to the disease because they carried CD22 tumor cells that the CD22 CAR T cells could not target. Treatment with three and eight million dual CAR T cells showed that while both treatments were effective, animals treated with CD20xCD22 unexpectedly survived longer than animals treated with CD22xCD20.

Example 9. Interferon gamma (IFN-gamma) release by dual CD20 and CD22 CAR-T cells

Daudi cells expressing CD20 and CD22 are engineered to express luciferase and GFP. These Daudi cells are incubated overnight with CD20xCD22, CD22xCD20, CD22, or CD20-positive CAR-T cells at a 1:1 ratio (effector:target). The next day, the plate is centrifuged and the supernatant is collected. The levels of interferon gamma released during tumor incubation with CAR-T cells are quantified using the Quantikine human interferon gamma ELISA kit (R&D systems, DIF50C) according to the manufacturer's instructions. As a positive control, CAR-T cells were incubated with phorbol myristate acetate (PMA; Sigma Aldrich, P8139) and ionomycin (Sigma Aldrich, 10634). The final IFN-gamma release values were normalized to those in the positive control. Figure 8 shows that dual CD20×CD22 CAR-T cells as well as CD22×CD20 CAR-T cells are activated upon recognition of antigen on tumor cells and release higher levels of IFN-gamma than single CAR-T cells (CD20 or CD22). This observation indicates a synergistic effect of the use of dual (CD20, CD22) CAR-T cells.

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SEQUENCE LIST

<110> SELLECTIS S.A.

<120> CAR T CELLS WITH TWO CHIMERIC ANTIGEN RECEPTORS

<130> P242859PC00

<150> PA202070509

<151> 2020-07-31

<160> 52

<170> PatentIn version 3.5

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<212> PRT

<213> artificial

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala Ala Arg Pro

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Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

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<212> PRT

<213> artificial

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Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

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<211> 231

<212> PRT

<213> artificial

<220>

<223> IgG1 hinge

<400> 5

Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 6

<211> 24

<212> PRT

<213> artificial

<220>

<223> CD8 alpha transmembrane domain

<400> 6

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 7

<211> 27

<212> PRT

<213> artificial

<220>

<223> 4-1BB transmembrane domain

<400> 7

Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu

1 5 10 15

Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val

20 25

<210> 8

<211> 42

<212> PRT

<213> artificial

<220>

<223> 4-1BB stimulatory domain

<400> 8

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 9

<211> 112

<212> PRT

<213> artificial

<220>

<223> CD3 zeta signaling domain

<400> 9

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 10

<211> 15

<212> PRT

<213> artificial

<220>

<223> Linker

<400> 10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 11

<211> 124

<212> PRT

<213> artificial

<220>

<223> CAR22 VH

<400> 11

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> PRT

<213> artificial

<220>

<223> CAR22 VL

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 246

<212> PRT

<213> artificial

<220>

<223> CAR22 scFv

<400> 13

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr

130 135 140

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

145 150 155 160

Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr Leu Asn Trp Tyr Gln

165 170 175

Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr Ala Ala Ser Ser

180 185 190

Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Arg Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Phe Ala Thr

210 215 220

Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln Thr Phe Gly Gln Gly

225 230 235 240

Th Lys Leu Glu Ile Lys

245

<210> 14

<211> 490

<212> PRT

<213> artificial

<220>

<223> CAR22 full

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp

35 40 45

Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro

50 55 60

Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp

65 70 75 80

Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro

85 90 95

Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu

115 120 125

Glu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

165 170 175

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr

180 185 190

Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile

195 200 205

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

225 230 235 240

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln

245 250 255

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 15

<211> 122

<212> PRT

<213> artificial

<220>

<223> CAR20 VH

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 107

<212> PRT

<213> artificial

<220>

<223> CAR20 VL

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 17

<211> 244

<212> PRT

<213> artificial

<220>

<223> CAR20scFv

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser

130 135 140

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala

180 185 190

Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr

210 215 220

Cys Gln Gln Arg Ser Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg

225 230 235 240

Leu Glu Ile Lys

<210> 18

<211> 488

<212> PRT

<213> artificial

<220>

<223> CAR20 full

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Lys Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Leu Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr

115 120 125

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

145 150 155 160

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

165 170 175

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp

195 200 205

Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp

225 230 235 240

Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile Thr Phe

245 250 255

Gly Gln Gly Thr Arg Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 19

<211> 22

<212> PRT

<213> artificial

<220>

<223> P2A peptide

<400> 19

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 20

<211> 317

<212> PRT

<213> artificial

<220>

<223> CD20-6His

<400> 20

Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro

1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg

20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu

35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile

50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile

65 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile

85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu

100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile

115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser

130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro

145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn

165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly

180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile

195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys

210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile

225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro

245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu

260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser

275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro Gly Gly Gly Ser Gly Gly Gly

290 295 300

Ser His His His His His His His His His His

305 310 315

<210> 21

<211> 916

<212> PRT

<213> artificial

<220>

<223> CD22-Fc

<400> 21

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Asp Ser Ser Lys Trp Val Phe Glu His Pro Glu Thr Leu

20 25 30

Tyr Ala Trp Glu Gly Ala Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala

35 40 45

Leu Asp Gly Asp Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr

50 55 60

Asn Lys Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr

65 70 75 80

Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly

85 90 95

Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu Asn

100 105 110

Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr Glu Lys Trp

115 120 125

Met Glu Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro His

130 135 140

Ile Gln Leu Pro Pro Glu Ile Gln Glu Ser Gln Glu Val Thr Leu Thr

145 150 155 160

Cys Leu Leu Asn Phe Ser Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp

165 170 175

Leu Leu Glu Gly Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser

180 185 190

Leu Thr Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro

195 200 205

Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala

210 215 220

Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys His

225 230 235 240

Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp Ala Ile Val Arg

245 250 255

Glu Gly Asp Ser Val Thr Met Thr Cys Glu Val Ser Ser Ser Asn Pro

260 265 270

Glu Tyr Thr Thr Val Ser Trp Leu Lys Asp Gly Thr Ser Leu Lys Lys

275 280 285

Gln Asn Thr Phe Thr Leu Asn Leu Arg Glu Val Thr Lys Asp Gln Ser

290 295 300

Gly Lys Tyr Cys Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser

305 310 315 320

Glu Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val

325 330 335

Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu

340 345 350

Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp Tyr His

355 360 365

Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys Val His Ile Pro

370 375 380

Lys Ile Leu Pro Trp His Ala Gly Thr Tyr Ser Cys Val Ala Glu Asn

385 390 395 400

Ile Leu Gly Thr Gly Gln Arg Gly Pro Gly Ala Glu Leu Asp Val Gln

405 410 415

Tyr Pro Pro Lys Lys Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile

420 425 430

Arg Glu Gly Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn

435 440 445

Pro Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu

450 455 460

Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr

465 470 475 480

Thr Ile Ala Cys Ala Ala Cys Asn Ser Trp Cys Ser Trp Ala Ser Pro

485 490 495

Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp Val Arg Val Arg Lys

500 505 510

Ile Lys Pro Leu Ser Glu Ile His Ser Gly Asn Ser Val Ser Leu Gln

515 520 525

Cys Asp Phe Ser Ser Ser His Pro Lys Glu Val Gln Phe Phe Trp Glu

530 535 540

Lys Asn Gly Arg Leu Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser

545 550 555 560

Ile Ser Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser

565 570 575

Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala

580 585 590

Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met Glu

595 600 605

Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn Pro Pro Val

610 615 620

Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln Ser Leu Pro Tyr His

625 630 635 640

Ser Gln Lys Leu Arg Leu Glu Pro Val Lys Val Gln His Ser Gly Ala

645 650 655

Tyr Trp Cys Gln Gly Thr Asn Ser Val Gly Lys Gly Arg Ser Pro Leu

660 665 670

Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Gly

675 680 685

Gly Gly Gly Ala Gly Gly Gly Gly Cys Lys Pro Cys Ile Cys Thr Val

690 695 700

Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val

705 710 715 720

Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile

725 730 735

Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val

740 745 750

Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser

755 760 765

Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu

770 775 780

Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala

785 790 795 800

Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro

805 810 815

Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Asp Lys Val

820 825 830

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

835 840 845

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

850 855 860

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

865 870 875 880

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

885 890 895

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

900 905 910

Ser Pro Gly Lys

915

<210> 22

<211> 9

<212> PRT

<213> artificial

<220>

<223> rituximab-specific epitope

<400> 22

Cys Pro Tyr Ser Asn Pro Ser Leu Cys

1 5

<210> 23

<211> 925

<212> PRT

<213> artificial

<220>

<223> TRAC_T01-L

<400> 23

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

210 215 220

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

245 250 255

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

515 520 525

Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 24

<211> 925

<212> PRT

<213> artificial

<220>

<223> TRAC_T01-R

<400> 24

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

145 150 155 160

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

210 215 220

Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

420 425 430

Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

515 520 525

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 25

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD52_T01-L

<400> 25

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile

180 185 190

Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

210 215 220

Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

485 490 495

Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

515 520 525

Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 26

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD52_T01-R

<400> 26

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val

210 215 220

Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile

450 455 460

Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

515 520 525

Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 27

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD22_T01-L

<400> 27

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

145 150 155 160

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

210 215 220

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

515 520 525

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 28

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD22_T01-R

<400> 28

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

210 215 220

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

515 520 525

Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 29

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD20_T01-L

<400> 29

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

210 215 220

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

245 250 255

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

420 425 430

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln

515 520 525

Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 30

<211> 925

<212> PRT

<213> artificial

<220>

<223> CD20_T01-R

<400> 30

Met Gly Asp Pro Lys Lys Lys Arg Lys Val Ile Asp Ile Ala Asp Leu

1 5 10 15

Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys

20 25 30

Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly

35 40 45

Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu

50 55 60

Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu

65 70 75 80

Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala

85 90 95

Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro

100 105 110

Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly

115 120 125

Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr

130 135 140

Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser

145 150 155 160

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro

165 170 175

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

180 185 190

Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

195 200 205

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

210 215 220

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

225 230 235 240

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

245 250 255

Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr

260 265 270

Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

275 280 285

Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu

290 295 300

Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

305 310 315 320

Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln

325 330 335

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His

340 345 350

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

355 360 365

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

370 375 380

Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile

385 390 395 400

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu

405 410 415

Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser

420 425 430

Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

435 440 445

Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile

450 455 460

Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

465 470 475 480

Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val

485 490 495

Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

500 505 510

Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln

515 520 525

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

530 535 540

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro

545 550 555 560

Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu

565 570 575

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu

580 585 590

Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln

595 600 605

Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His

610 615 620

Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly

625 630 635 640

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

645 650 655

Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly

660 665 670

Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln Leu Ser Arg Pro

675 680 685

Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val Ala Leu Ala

690 695 700

Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys Lys Gly Leu Gly

705 710 715 720

Asp Pro Ile Ser Arg Ser Gln Leu Val Lys Ser Glu Leu Glu Glu Lys

725 730 735

Lys Ser Glu Leu Arg His Lys Leu Lys Tyr Val Pro His Glu Tyr Ile

740 745 750

Glu Leu Ile Glu Ile Ala Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu

755 760 765

Met Lys Val Met Glu Phe Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys

770 775 780

His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly

785 790 795 800

Ser Pro Ile Asp Tyr Gly Val Ile Val Asp Thr Lys Ala Tyr Ser Gly

805 810 815

Gly Tyr Asn Leu Pro Ile Gly Gln Ala Asp Glu Met Gln Arg Tyr Val

820 825 830

Glu Glu Asn Gln Thr Arg Asn Lys His Ile Asn Pro Asn Glu Trp Trp

835 840 845

Lys Val Tyr Pro Ser Ser Val Thr Glu Phe Lys Phe Leu Phe Val Ser

850 855 860

Gly His Phe Lys Gly Asn Tyr Lys Ala Gln Leu Thr Arg Leu Asn His

865 870 875 880

Ile Thr Asn Cys Asn Gly Ala Val Leu Ser Val Glu Glu Leu Leu Ile

885 890 895

Gly Gly Glu Met Ile Lys Ala Gly Thr Leu Thr Leu Glu Glu Val Arg

900 905 910

Arg Lys Phe Asn Asn Gly Glu Ile Asn Phe Ala Ala Asp

915 920 925

<210> 31

<211> 3003

<212> DNA

<213> artificial

<220>

<223> nt seq CAR22xCAR20

<400> 31

atggctctgc ccgtcaccgc tctgctgctg ccactggccc tgctgctgca cgcagcaaga 60

ccacaggtgc agctgcagca gagcggccct ggcctggtga agccaagcca gacactgtcc 120

ctgacctgcg ccatcagcgg cgattccgtg agctccaact ccgccgcctg gaattggatc 180

aggcagtccc cttctcgggg cctggagtgg ctgggaagga catactatcg gtctaagtgg 240

tacaacgatt atgccgtgtc tgtgaagagc agaatcacaa tcaaccctga cacctccaag 300

aatcagttct ctctgcagct gaatagcgtg acaccagagg acaccgccgt gtactattgc 360

gccagggagg tgaccggcga cctggaggat gcctttgaca tctggggcca gggcacaatg 420

gtgaccgtgt ctagcggagg aggaggatcc ggaggaggag gatctggcgg cggcggcagc 480

gatatccaga tgacacagtc cccatcctct ctgagcgcct ccgtgggcga cagagtgaca 540

atcacctgta gggcctccca gaccatctgg tcttacctga actggtatca gcagaggccc 600

ggcaaggccc ctaatctgct gatctacgca gcaagctccc tgcagagcgg agtgccatcc 660

agattctctg gcaggggctc cggcacagac ttcaccctga ccatctctag cctgcaggcc 720

gaggacttcg ccacctacta ttgccagcag tcttatagca tcccccagac atttggccag 780

ggcaccaagc tggagatcaa gaccacaacc ccagcaccaa ggccacctac acctgcacca 840

accatcgcct ctcagcccct gagcctgaga cctgaggcat gtaggccagc agcaggagga 900

gcagtccata caaggggtct ggattttgca tgcgacatct acatctgggc acctctggca 960

ggaacatgtg gcgtgctcct gctcagcctg gtcatcaccc tgtactgcaa gagaggcagg 1020

aagaagctgc tgtatatctt caagcagccc ttcatgcgcc ccgtgcagac aacccaggag 1080

gaggatggct gctcctgtag gttcccagaa gaggaggagg gaggatgtga gctgcgcgtg 1140

aagttttccc ggtctgccga cgcacctgca taccagcagg gccagaacca gctgtataac 1200

gagctgaatc tgggccggag agaggagtac gatgtgctgg acaagaggcg cggcagagat 1260

ccagagatgg gcggcaagcc ccggagaaag aaccctcagg agggcctgta caatgagctg 1320

cagaaggata agatggccga ggcctattct gagatcggca tgaagggaga gaggcgccgg 1380

ggcaagggac acgacggact gtaccaggga ctgagcacag ccaccaagga tacctatgac 1440

gccctgcata tgcaggcact gcctccaagg ggaagcggag ctactaactt cagcctgctg 1500

aagcaggctg gagacgtgga ggagaaccct ggacctatgg ctctgcctgt caccgctctg 1560

ctgctgcccc tggctctgct gctgcacgcc gcacgccccg aagtccagct ggtcgaatct 1620

gggggcgggc tggtgcagcc aggaagatca ctgaggctga gctgcgccgc ttccggcttc 1680

accttcaacg actatgccat gcactgggtg agacaggctc ccggaaaggg cctggagtgg 1740

gtctctacca tcagttggaa ttccgggtct attggatatg ccgacagcgt gaaaggccgc 1800

ttcacaatct ctcgagataa cgctaagaaa agtctgtacc tgcagatgaa ttcactgagg 1860

gcagaggaca ctgccctgta ctattgcgcc aaggatattc agtacggcaa ctactattac 1920

gggatggacg tctggggggca gggaaccaca gtgaccgtca gctccggagg aggaggatcc 1980

ggaggaggag gaagcggagg aggaggatcc gagatcgtgc tgacacagag cccagccact 2040

ctgagtctgt cacccggcga acgagctaca ctgtcctgtc gggcaagcca gtccgtctct 2100

agttatctgg cttggtacca gcagaagcca ggacaggcac cacgactgct gatctacgat 2160

gctagcaaca gagcaacagg gattcctgca aggttctctg gcagtgggtc aggaactgac 2220

tttacactga ctatctcaag cctggagcct gaagatttcg ccgtgtatta ctgccagcag 2280

cggtccaatt ggccaatcac ctttggccag gggacacgcc tggagatcaa gactaccaca 2340

cctgctccac gaccacctac tccagcacct accattgctt ctcagcccct gagtctgcgg 2400

cctgaagcat gtagaccagc agcaggagga gcagtgcata ccagaggact ggactttgcc 2460

tgcgatatct atatttgggc accactggct ggaacttgtg gagtgctgct gctgtctctg 2520

gtcatcaccc tgtattgcaa gcgaggccgg aagaaactgc tgtacatttt caaacagcct 2580

tttatgagac cagtgcagac tacccaggag gaagacggct gcagctgtag gttccccgag 2640

gaagaggaag ggggatgtga gctgagggtc aagtttagcc gctccgctga tgcacctgcc 2700

tatcagcagg ggcagaatca gctgtacaac gagctgaatc tgggacggag agaggaatac 2760

gacgtgctgg ataaaaggcg aggacgagat ccagaaatgg gagggaagcc ccgacggaaa 2820

aaccctcagg agggcctgta taatgaactg cagaaggaca aaatggctga ggcatactct 2880

gaaatcggaa tgaagggcga gagaaggcgc ggaaaaggcc acgatgggct gtatcaggga 2940

ctgagtaccg ccacaaagga cacctacgat gcactgcata tgcaggccct gccaccccgg 3000

tga 3003

<210> 32

<211> 3003

<212> DNA

<213> artificial

<220>

<223> nt seq CAR20xCAR22

<400> 32

atggctctgc ctgtcaccgc tctgctgctg cccctggctc tgctgctgca cgccgcacgc 60

cccgaagtcc agctggtcga atctgggggc gggctggtgc agccaggaag atcactgagg 120

ctgagctgcg ccgcttccgg cttcaccttc aacgactatg ccatgcactg ggtgagacag 180

gctcccggaa agggcctgga gtgggtctct accatcagtt ggaattccgg gtctattgga 240

tatgccgaca gcgtgaaagg ccgcttcaca atctctcgag ataacgctaa gaaaagtctg 300

tacctgcaga tgaattcact gagggcagag gacactgccc tgtactattg cgccaaggat 360

attcagtacg gcaactacta ttacgggatg gacgtctggg ggcagggaac cacagtgacc 420

gtcagctccg gaggaggagg atccggagga ggaggaagcg gaggaggagg atccgagatc 480

gtgctgacac agagcccagc cactctgagt ctgtcacccg gcgaacgagc tacactgtcc 540

tgtcgggcaa gccagtccgt ctctagttat ctggcttggt accagcagaa gccaggacag 600

gcaccacgac tgctgatcta cgatgctagc aacagagcaa cagggattcc tgcaaggttc 660

tctggcagtg ggtcaggaac tgactttaca ctgactatct caagcctgga gcctgaagat 720

ttcgccgtgt attactgcca gcagcggtcc aattggccaa tcacctttgg ccaggggaca 780

cgcctggaga tcaagactac cacacctgct ccacgaccac ctactccagc acctaccatt 840

gcttctcagc ccctgagtct gcggcctgaa gcatgtagac cagcagcagg aggagcagtg 900

cataccagag gactggactt tgcctgcgat atctatattt gggcaccact ggctggaact 960

tgtggagtgc tgctgctgtc tctggtcatc accctgtatt gcaagcgagg ccggaagaaa 1020

ctgctgtaca ttttcaaaca gccttttatg agaccagtgc agactaccca ggaggaagac 1080

ggctgcagct gtaggttccc cgaggaagag gaagggggat gtgagctgag ggtcaagttt 1140

agccgctccg ctgatgcacc tgcctatcag caggggcaga atcagctgta caacgagctg 1200

aatctgggac ggagagagga atacgacgtg ctggataaaa ggcgaggacg agatccagaa 1260

atgggaggga agccccgacg gaaaaaccct caggagggcc tgtataatga actgcagaag 1320

gacaaaatgg ctgaggcata ctctgaaatc ggaatgaagg gcgagagaag gcgcggaaaa 1380

ggccacgatg ggctgtatca gggactgagt accgccacaa aggacaccta cgatgcactg 1440

catatgcagg ccctgccacc ccgaggaagc ggagctacta acttcagcct gctgaagcag 1500

gctggagacg tggaggagaa ccctggacct atggctctgc ccgtcaccgc tctgctgctg 1560

ccactggccc tgctgctgca cgcagcaaga ccacaggtgc agctgcagca gagcggccct 1620

ggcctggtga agccaagcca gacactgtcc ctgacctgcg ccatcagcgg cgattccgtg 1680

agctccaact ccgccgcctg gaattggatc aggcagtccc cttctcgggg cctggagtgg 1740

ctgggaagga catactatcg gtctaagtgg tacaacgatt atgccgtgtc tgtgaagagc 1800

agaatcacaa tcaaccctga cacctccaag aatcagttct ctctgcagct gaatagcgtg 1860

acaccagagg acaccgccgt gtactattgc gccagggagg tgaccggcga cctggaggat 1920

gcctttgaca tctggggcca gggcacaatg gtgaccgtgt ctagcggagg aggaggatcc 1980

ggaggaggag gatctggcgg cggcggcagc gatatccaga tgacacagtc cccatcctct 2040

ctgagcgcct ccgtgggcga cagagtgaca atcacctgta gggcctccca gaccatctgg 2100

tcttacctga actggtatca gcagaggccc ggcaaggccc ctaatctgct gatctacgca 2160

gcaagctccc tgcagagcgg agtgccatcc agattctctg gcaggggctc cggcacagac 2220

ttcaccctga ccatctctag cctgcaggcc gaggacttcg ccacctacta ttgccagcag 2280

tcttatagca tcccccagac atttggccag ggcaccaagc tggagatcaa gaccacaacc 2340

ccagcaccaa ggccacctac acctgcacca accatcgcct ctcagcccct gagcctgaga 2400

cctgaggcat gtaggccagc agcaggagga gcagtccata caaggggtct ggattttgca 2460

tgcgacatct acatctgggc acctctggca ggaacatgtg gcgtgctcct gctcagcctg 2520

gtcatcaccc tgtactgcaa gagaggcagg aagaagctgc tgtatatctt caagcagccc 2580

ttcatgcgcc ccgtgcagac aacccaggag gaggatggct gctcctgtag gttcccagaa 2640

gaggaggagg gaggatgtga gctgcgcgtg aagttttccc ggtctgccga cgcacctgca 2700

taccagcagg gccagaacca gctgtataac gagctgaatc tgggccggag agaggagtac 2760

gatgtgctgg acaagaggcg cggcagagat ccagagatgg gcggcaagcc ccggagaaag 2820

aaccctcagg agggcctgta caatgagctg cagaaggata agatggccga ggcctattct 2880

gagatcggca tgaagggaga gaggcgccgg ggcaagggac acgacggact gtaccaggga 2940

ctgagcacag ccaccaagga tacctatgac gccctgcata tgcaggcact gcctccaagg 3000

tga 3003

<210> 33

<211> 49

<212> DNA

<213> artificial

<220>

<223> TRAC_T01-target

<400> 33

ttgtcccaca gatatccaga accctgaccc tgccgtgtac cagctgaga 49

<210> 34

<211> 49

<212> DNA

<213> artificial

<220>

<223> CD52_T01-target

<400> 34

ttcctcctac tcaccatcag cctcctggtt atggtacagg taagagcaa 49

<210> 35

<211> 49

<212> DNA

<213> artificial

<220>

<223> CD22_T01 target

<400> 35

tctggttttc ttccagatcc tcccaagaag gtgaccacag tgattcaaa 49

<210> 36

<211> 49

<212> DNA

<213> artificial

<220>

<223> CD20_T01 target

<400> 36

tttgctgcca tttctggaat gattctttca atcatggaca tacttaata 49

<210> 37

<211> 588

<212> DNA

<213> artificial

<220>

<223> IRES

<400> 37

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacc 588

<210> 38

<211> 21

<212> PRT

<213> artificial

<220>

<223> T2A peptide

<400> 38

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 39

<211> 23

<212> PRT

<213> artificial

<220>

<223> E2A peptide

<400> 39

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 40

<211> 25

<212> PRT

<213> artificial

<220>

<223> F2A peptide

<400> 40

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 41

<211> 11

<212> PRT

<213> artificial

<220>

<223> CAR22 H-CDR1

<400> 41

Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Ala

1 5 10

<210> 42

<211> 9

<212> PRT

<213> artificial

<220>

<223> CAR22 H-CDR2

<400> 42

Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn

1 5

<210> 43

<211> 13

<212> PRT

<213> artificial

<220>

<223> CAR22 H-CDR3

<400> 43

Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp

1 5 10

<210> 44

<211> 6

<212> PRT

<213> artificial

<220>

<223> CAR22 L-CDR1

<400> 44

Gln Thr Ile Trp Ser Tyr

1 5

<210> 45

<211> 3

<212> PRT

<213> artificial

<220>

<223> CAR22 L-CDR2

<400> 45

Ala Ala Ser

1

<210> 46

<211> 9

<212> PRT

<213> artificial

<220>

<223> CAR22 L-CDR3

<400> 46

Gln Gln Ser Tyr Ser Ile Pro Gln Thr

1 5

<210> 47

<211> 8

<212> PRT

<213> artificial

<220>

<223> CAR20 H-CDR1

<400> 47

Gly Phe Thr Phe Asn Asp Tyr Ala

1 5

<210> 48

<211> 8

<212> PRT

<213> artificial

<220>

<223> CAR20 H-CDR2

<400> 48

Ile Ser Trp Asn Ser Gly Ser Ile

1 5

<210> 49

<211> 15

<212> PRT

<213> artificial

<220>

<223> CAR20 H-CDR3

<400> 49

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 50

<211> 6

<212> PRT

<213> artificial

<220>

<223> CAR20 L-CDR1

<400> 50

Gln Ser Val Ser Ser Tyr

1 5

<210> 51

<211> 3

<212> PRT

<213> artificial

<220>

<223> CAR20 L-CDR2

<400> 51

Asp Ala Ser

1

<210> 52

<211> 9

<212> PRT

<213> artificial

<220>

<223> CAR20 L-CDR3

<400> 52

Gln Gln Arg Ser Asn Trp Pro Ile Thr

1 5

<---

Claims (48)

1. A genetically engineered T cell expressing on its surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20), a) where the specified CAR22 includes: i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 11, and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 12, and optionally a leader sequence, - hinge domain of CD8alpha, ii) the transmembrane domain of CD8alpha, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; and b) where the specified CAR20 includes: i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 15, and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 16, and optionally a leader sequence, - hinge domain of CD8alpha, ii) the transmembrane domain of CD8alpha, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; wherein said CAR22 and said CAR20 are encoded by an exogenous nucleic acid integrated into the genome of said T cell; wherein said exogenous nucleic acid comprises in the 5' to 3' direction: (i) promoter, (ii) a nucleic acid encoding said CAR20, (iii) a nucleic acid encoding a self-cleaving peptide; (iv) a nucleic acid encoding said CAR22, where the same promoter controls the expression of these CAR20 and CAR22. 2. The genetically engineered T cell of claim 1, wherein said CAR22 comprises the amino acid sequence of SEQ ID NO: 14 and said CAR20 comprises the amino acid sequence of SEQ ID NO: 18. 3. The genetically engineered T cell of any one of claims 1 and 2, wherein said exogenous nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 32. 4. A genetically engineered T cell according to any one of claims 1-3, wherein said engineered T cell is a T cell that is TCR negative. 5. A genetically engineered T cell according to claim 4, wherein said engineered T cell comprises at least one allele encoding TCR alpha, TCR beta and/or CD3 inactivated by mutation. 6. A genetically engineered T cell according to any one of claims 4, 5, wherein said engineered T cell comprises at least one allele selected from β2m, PD1, CTLA4, dCK, CD52 and/or GR that has been inactivated. 7. A pharmaceutical composition for the treatment of cancer associated with the expression of CD20 and CD22, comprising engineered T cells according to any one of claims 1-6 and a pharmaceutically acceptable excipient. 8. A pharmaceutical composition for the treatment of cancer associated with CD20 expression, comprising engineered T cells according to any one of claims 1-6 and a pharmaceutically acceptable excipient. 9. A pharmaceutical composition for the treatment of cancer associated with CD22 expression, comprising engineered T cells according to any one of claims 1-6 and a pharmaceutically acceptable excipient. 10. An isolated polynucleotide encoding CAR20 and CAR22, which comprises: a) a promoter that controls the expression of CAR20, followed by a nucleic acid encoding said CAR20, wherein said CAR20 comprises: i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 15, and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 16, and optionally a leader sequence, - hinge domain of CD8alpha, ii) the transmembrane domain of CD8alpha, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; b) a nucleic acid encoding CAR22, wherein said CAR22 comprises: i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 11, and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 12, and optionally a leader sequence, - hinge domain of CD8alpha, ii) the transmembrane domain of CD8alpha, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3zeta signaling domain; wherein said nucleic acids a) and b) are located in one nucleic acid molecule, and wherein the nucleic acid sequence encoding the self-cleaving peptide is located between said nucleic acids a) and b). 11. The isolated polynucleotide according to claim 10, containing the nucleic acid sequence SEQ ID NO: 32. 12. An expression vector containing an isolated polynucleotide according to any one of paragraphs 10, 11. 13. A method for producing an engineered T cell according to any one of claims 1-6, comprising introducing into the T cell a polynucleotide according to any one of claims 10-11 or a vector according to claim 12. 14. Use of an engineered T cell according to any one of claims 1-6 for the treatment of cancer associated with the expression of CD20 and CD22. 15. Use of an engineered T cell according to any one of claims 1-6 for the treatment of cancer associated with CD20 expression. 16. Use of an engineered T cell according to any one of claims 1-6 for the treatment of cancer associated with CD22 expression. 17. The use of an engineered T cell according to any one of claims 14-16, wherein said cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphocytic cancer, acute myeloid leukemia (AML).