EA043737B1 - COMPOSITIONS AND METHODS FOR REPROGRAMMING T-CELL RECEPTORS USING HYBRID PROTEINS - Google Patents

Description

Cross reference

This application claims priority to U.S. Provisional Patent Application No. 62/405,551, filed Oct. 7, 2016, and U.S. Provisional Patent Application No. 62/510,108, filed May 23.

2017, each of which is incorporated herein by reference in its entirety.

State of the art

Most patients with advanced solid tumors are not curable with standard therapy. In addition, traditional treatments often have serious side effects. Numerous attempts have been made to harness the patient's immune system to reject cancer cells, an approach collectively referred to as cancer immunotherapy. However, due to several obstacles, achieving clinical effectiveness becomes quite difficult. Although hundreds of so-called tumor antigens have been identified, they are often self-specific and can therefore direct the action of cancer immunotherapy against healthy tissue, or they are weakly immunogenic. Moreover, cancer cells use multiple mechanisms to become invisible or hostile to the initiation and propagation of immune attack during cancer immunotherapy.

Recent developments that use autologous chimeric antigen receptor (CAR)-modified T cell therapy, which relies on redirecting genetically engineered T cells to the appropriate cell surface molecule on cancer cells, are showing promise in terms of harnessing the immune system's capabilities to treatment of B-cell malignancies (see, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results for CD-19-specific CAR-T cells (called CTL019) have demonstrated complete remission in patients suffering from chronic lymphocytic leukemia (CLL), as well as in children with acute lymphoblastic leukemia (ALL) (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). An alternative approach is to use T-cell receptor (TCR) alpha and beta chains selected for tumor-associated peptide antigen for genetically engineered autologous T cells. Such TCR chains will form complete TCR complexes and provide TCR T cells with a second defined specificity. Promising results were obtained with engineered autologous T cells expressing NY-ESO-1-specific TCR alpha and beta chains in patients with synovial carcinoma.

In addition to the ability of genetically engineered T cells expressing a CAR or second TCR to recognize and kill appropriate target cells in vitro/ex vivo, successful therapy for a patient using engineered T cells requires the ability of T cells to be strongly activated, proliferate, persist with over time, and in case of relapse of the disease - to ensure a secondary response. The high and controlled clinical efficacy of CAR-T cells is currently limited to BCMA- and CD-19-positive B-cell malignancies and patients with HLA-A2-expressing NY-ESO-1-peptide-expressing synovial sarcomas. There is a clear need to improve genetically engineered T cells for broader action against a variety of human malignancies. Herein, we describe novel fusion proteins of TCR subunits, including CD3 epsilon, CD3 gamma, CD3 delta, and TCR alpha and beta chains, with binding domains specific for cell surface antigens, which may potentially overcome the limitations of existing approaches. Described herein are novel fusion proteins that are more effective at killing target cells than CARs but release comparable or lower levels of pro-inflammatory cytokines. These fusion proteins and their uses represent an advantage of T-cell receptor (TCR) fusion proteins (TFPs) over CARs because elevated levels of such cytokines are associated with dose-limiting toxicities of adoptive CAR-T therapy.

The essence of the invention

Provided herein are T cell receptor (TCR) fusion proteins (TFPs), T cells engineered to express one or more TFPs, and methods of using them to treat diseases.

In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit and a human or humanized antibody domain comprising an anti-mesothelin binding domain.

In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain. CD3 epsilon domain; and a human or humanized antibody domain comprising an antigen binding domain, wherein the TCR subunit and the antibody domain are operably linked, and wherein TFP is incorporated into the TCR

- 1 043737 when expressed in a T cell.

In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain. CD3 gamma domain; and a human or humanized antibody domain comprising an antigen binding domain, wherein the TCR subunit and the antibody domain are operably linked and wherein TFP is incorporated into the TCR when expressed in a T cell. In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain. CD3 delta domain; and a human or humanized antibody domain comprising an antigen binding domain, wherein the TCR subunit and the antibody domain are operably linked and wherein TFP is incorporated into the TCR when expressed in a T cell. In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a Tcellular receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from a TCR intracellular signaling domain. alpha; and a human or humanized antibody domain comprising an antigen binding domain, wherein the TCR subunit and the antibody domain are operably linked and wherein TFP is incorporated into the TCR when expressed in a T cell. In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain. TCR beta domain; and a human or humanized antibody domain comprising an antigen binding domain, wherein the TCR subunit and the antibody domain are operably linked and wherein TFP is incorporated into the TCR when expressed in a T cell. In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit and a human or humanized antibody domain containing an antigen binding domain that is an anti-mesothelin binding domain.

In some cases, the TCR subunit and the antibody domain are operably linked. In some cases, TFP is inserted into the TCR when expressed in a T cell. In some cases, the encoded antigen binding domain is connected to the extracellular domain of the TCR via a linker sequence. In some cases, the encoded linker sequence contains (G ₄ S) _n , where n = 1 to 4. In some cases, the TCR subunit contains a TCR extracellular domain. In some cases, the TCR subunit contains a TCR transmembrane domain. In some cases, the TCR subunit contains an intracellular TCR domain. In some cases, a TCR subunit comprises (i) an extracellular TCR domain, (ii) a transmembrane TCR domain, and (iii) an intracellular TCR domain, where at least two of (i), (ii), and (iii) are from the same TCR subunit. In some cases, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from a CD3 epsilon, CD3 gamma or CD3 delta intracellular signaling domain, or an amino acid sequence further having at least one, two or three modifications. In some cases, the TCR subunit contains an intracellular domain containing a stimulatory domain selected from a functional 4-1BB signaling domain and/or a functional CD3 zeta signaling domain, or an amino acid sequence further having at least one modification. In some cases, the domain of a human or humanized antibody contains an antibody fragment. In some cases, the domain of a human or humanized antibody contains a scFv or a _VH domain. In some cases, the isolated nucleic acid molecule encodes (i) light chain (LC) CDR1, LC CDR2, and LC CDR3 of the anti-mesothelin light chain binding domain amino acid sequence with 70-100% sequence identity to light chain (LC) CDR1, LC CDR2, and LC CDR3 LC of the anti-mesothelin light chain binding domain provided herein, respectively, and/or (ii) CDR1 heavy chain (HC), CDR2 HC, and CDR3 HC amino acid sequence of the anti-mesothelin heavy chain binding domain with 70-100% sequence identity to CDR1 heavy chain (HC), CDR2 HC and CDR3 HC of the anti-mesothelin heavy chain binding domain provided herein, respectively. In some cases, the isolated nucleic acid molecule encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one, but not more than 30 light chain variable region amino acid sequence modifications for the light chain variable region provided herein, or a sequence with 95-99% identity to the light chain variable region amino acid sequence for the light chain variable region provided herein. In some cases, the isolated nucleic acid molecule encodes a heavy chain variable region, the variable

- 2 043737 the heavy chain variable region contains an amino acid sequence having at least one, but not more than 30 modifications of the heavy chain variable region amino acid sequence for the heavy chain variable region presented herein, or a sequence with 95-99% identity to the amino acid sequence heavy chain variable region for the heavy chain variable region provided herein. In some cases, the TFP includes an extracellular domain of a TCR subunit that contains an extracellular domain, or a portion thereof, of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, TCR subunit CD3 delta, their functional fragments and their amino acid sequences having at least one, but not more than 20 modifications. In some cases, the encoded TFP includes a transmembrane domain that contains a transmembrane domain of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, TCR CD3 delta subunit, functional ones thereof fragments and their amino acid sequences having at least one, but not more than 20 modifications. In some cases, the encoded TFP includes a transmembrane domain that contains a transmembrane domain of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, TCR subunit CD3 delta, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof and amino acid sequences thereof having at least one , but no more than 20 modifications. In some cases, the isolated nucleic acid molecule further contains a sequence encoding a co-stimulatory domain. In some cases, the co-stimulatory domain is a functional signaling domain derived from a protein selected from the group consisting of DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18 ), ICOS (CD278) and 4-1BB (CD137), as well as their amino acid sequences, which also have at least one, but not more than 20 modifications. In some cases, the isolated nucleic acid molecule additionally contains a leader sequence. In some cases, the isolated nucleic acid molecule is mRNA.

In some cases, the TFP includes an immunoreceptor tyrosine activating motif (ITAM) of a TCR subunit that contains an ITAM or a portion of a protein thereof selected from the group consisting of TCR CD3 zeta subunit, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, TCR CD3 delta subunit , TCR zeta chain, Fc epsilon receptor chain 1, Fc epsilon receptor chain 2, Fc gamma receptor chain 1, Fc gamma receptor chain 2a, Fc gamma receptor chain 2b1, Fc gamma receptor chain 2b2, Fc gamma receptor chains 3a, Fc gamma receptor chains 3b, Fc beta receptor chains 1, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278 , CD66d, their functional fragments and their amino acid sequences, also having at least one, but not more than 20 modifications. In some cases, ITAM replaces ITAM CD3 gamma, CD3 delta, or CD3 epsilon. In some cases, the ITAM is selected from the group consisting of TCR CD3 zeta subunit, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, and TCR CD3 delta subunit, and replaces another ITAM selected from the group consisting of TCR CD3 zeta subunit, TCR CD3 epsilon subunit , TCR CD3 gamma subunits and TCR CD3 delta subunits. In some cases, the nucleic acid contains a nucleotide analogue. In some cases, the nucleotide analogue is selected from the group consisting of 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2 '-fluorine, 2'-O-aminopropyl (2'-O-AP), 2'-Odimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-Odimethylaminoethyloxyethyl (2-O-DMAEOE), modified 2-O-N-methylacetamido (2-O-NMA), locked nucleic acid (GNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1,5-hydrohexitol nucleic acid (ANA), morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide and 2'-fluoro N3-P5'-phosphoramidite. In one aspect, provided herein is an isolated polypeptide molecule encoded by a nucleic acid molecule provided herein. In one aspect, provided herein is an isolated TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In one aspect, provided herein is an isolated TFP molecule comprising a human or humanized anti-mesothelin binding domain, an extracellular TCR domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functional interaction with an endogenous TCR complex and/or at least one endogenous TCR complex. TCR polypeptide.

In some embodiments, a domain of a human or humanized antibody comprising an antigen binding domain that is a nucleic acid-encoded anti-mesothelin binding domain, or an antibody comprising an anti-mesothelin binding domain, or a cell expressing a nucleic acid-encoded anti-mesothelin binding domain is affinities maximum around 200 nM, 100 nM, 75 nM, and 50 nM, 25 nM,

- 3 043737 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0. 02 nM or 0.01 nM; and/or at least about 100 nM, 75 nM, and 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM , 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM; and/or about 200 nM, 100 nM, 75 nM, and 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM , 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM.

In one aspect, provided herein is an isolated TFP molecule comprising a human or humanized anti-mesothelin binding domain, an extracellular TCR domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functional integration into the endogenous TCR complex. In some cases, the isolated TFP molecule contains an antibody or antibody fragment comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some cases, the anti-mesothelin binding domain is a scFv, a VH domain, or a camelid VHH domain. In some cases, the anti-mesothelin binding domain comprises a heavy chain with 95-100% identity to the heavy chain amino acid sequence provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some cases, the anti-mesothelin binding domain comprises a light chain with 95-100% identity to the light chain amino acid sequence provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some cases, the isolated TFP molecule contains a TCR extracellular domain that contains an extracellular domain, or a portion thereof, of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR CD3 epsilon subunit, TCR CD3 gamma subunit, TCR CD3 delta subunit, their functional fragments and their amino acid sequences having at least one, but not more than 20 modifications. In some cases, the anti-mesothelin binding domain is connected to the extracellular domain of the TCR via a linker sequence. In some cases, the linker region contains (G ₄ S) _n , where n = 1 to 4.

In some cases, the isolated TFP molecule additionally contains a sequence encoding a co-stimulatory domain. In some cases, the isolated TFP molecule additionally contains a sequence encoding an intracellular signaling domain. In some cases, the isolated TFP molecule additionally contains a leader sequence.

In one aspect, provided herein is a vector comprising a nucleic acid molecule encoding the TFP provided herein. In some cases, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, Rous Sarcoma Virus (RSV) vector, or retroviral vector. In some cases, the vector additionally contains a promoter. In some cases, the vector is an in vitro transcribed vector. In some cases, the nucleic acid sequence in the vector additionally contains a poly(A) tail. In some cases, the nucleic acid sequence in the vector additionally contains a 3' untranslated region (3'UTR).

In one aspect, provided herein is a cell containing a vector provided herein. In some cases, the cell is a human T cell. In some cases, the T cell is a CD8+ or CD4+ T cell. In some cases, the T cell is a gamma delta T cell. In some cases, the T cell is an NK T cell. In some cases, the cell further contains a nucleic acid encoding an inhibitory molecule that contains a first polypeptide that contains at least a portion of the inhibitory molecule linked to a second polypeptide that contains a positive signal from an intracellular signaling domain. In some cases, the inhibitory molecule comprises a first polypeptide that contains at least a portion of PD1, and a second polypeptide that contains a co-stimulatory domain and a primary signaling domain.

In one aspect, provided herein is a human CD8+ or CD4+ T cell comprising at least two TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functional interaction with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, on, and/or on the surface of a human CD8+ or CD4+ T cell.

In one aspect, provided herein is a protein complex comprising: a TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR complex.

- 4 043737

In some cases, the TCR contains an extracellular domain, or a portion thereof, of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR CD3 epsilon subunit, TCR subunit

TCR CD3 gamma and TCR CD3 delta subunits. In some cases, the anti-mesothelin binding domain is connected to the extracellular domain of the TCR via a linker sequence. In some cases, the linker region contains (G ₄ S) _n , where n = 1 to 4.

In one aspect, provided herein is a human CD8+ or CD4+ T cell comprising at least two different TFP proteins per protein complex provided herein.

In one aspect, provided herein is a method for generating a cell, comprising transducing a T cell with a vector provided herein.

In one aspect, provided herein is a method of producing a population of cells containing engineered RNAs, comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA contains a nucleic acid encoding a TFP molecule provided herein.

In one aspect, provided herein is a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell expressing a TFP molecule provided herein or expressing a polypeptide molecule provided herein.

In some cases, the cell is an autologous T cell. In some cases, the cell is an allogeneic T cell. In some cases, the mammal is a human.

In one aspect, provided herein is a method of treating a mammal with a mesothelin expression disorder, comprising administering to the mammal an effective amount of a TFP molecule provided herein, a cell provided herein, or a polypeptide molecule provided herein. In some cases, the disease associated with mesothelin expression is selected from the group consisting of a proliferative disease, cancer, malignancy, a non-cancer indication associated with mesothelin expression. In some cases, the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureteral cancer, kidney cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, thymus cancer, cholangiocarcinoma and stomach cancer.

In some cases, the disease is cancer. In some cases, the disease is selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed ovarian Müllerian carcinoma, endometrioid mucinous ovarian carcinoma, pleural malignancy, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, uterine serous carcinoma, adenocarcinomas s lungs , extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, mammary adenocarcinoma, mesothelin expression disease, mesothelin expression disease, nonmucinating ovarian carcinoma, invasive ductal adenocarcinoma, lung adenocarcinoma, gastric adenocarcinoma /esophagus, colorectal adenocarcinoma, leukemia, childhood acute myeloid leukemia, invasive intraductal papillary mucinous tumor (IPMN), endometrial adenocarcinoma, gastric/esophageal adenocarcinoma, lung adenocarcinoma, mammary adenocarcinoma, and combinations thereof.

In some cases, cells expressing the TFP molecule are administered in combination with a substance that increases the effectiveness of the cell expressing the TFP molecule. In some cases, the mammal releases fewer cytokines compared to a mammal that is administered an effective number of anti-mesothelin chimeric antigen receptor (CAR) T cells. In some cases, cells expressing a TFP molecule are administered in combination with a substance that alleviates one or more side effects associated with administration of a cell expressing a TFP molecule. In some cases, cells expressing the TFP molecule are administered in combination with a substance that treats a disease associated with mesothelin.

In one aspect, the drug used is an isolated nucleic acid molecule as provided herein, an isolated polypeptide molecule as provided herein, an isolated TFP as provided herein, a complex as provided herein, a vector as provided herein, or cell presented herein.

In one aspect, provided herein is a method of treating a mammal with a mesothelin expression disorder, comprising administering to the mammal an effective amount of a TFP molecule provided herein, a cell provided herein, or a polypeptide molecule provided herein. herein, wherein the mammal releases fewer cytokines compared to a mammal administered an effective amount of T cells expressing anti-mesothelin chimeric antigen receptor (CAR).

Incorporation by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and separately identified for inclusion by reference.

Brief description of graphic materials

The new properties of the invention are described in detail in the accompanying claims. A better understanding of the features and advantages of the present invention will be obtained from the following detailed description, which sets forth illustrative embodiments employing the principles of the invention and the accompanying drawings in which:

In fig. 1 is a schematic illustration demonstrating the use of T cell receptor fusion polypeptides (TFPs) of the present invention. An example TFP contains an anti-mesothelin scFv and a full-length CD3 epsilon polypeptide fused via a (G ₄ S) ₃ linker sequence. When TFP is produced by or introduced into a T cell, it binds to other endogenous T cell receptor (TCR) polypeptides (shown as including two CD3 epsilon polypeptides, one CD3 gamma polypeptide, one CD3 delta polypeptide, two CD3 polypeptides zeta, one TCR alpha subunit and one TCR beta subunit, with the horizontal gray segment representing the plasma membrane) to generate a reprogrammed TCR in which one or both endogenous CD3 epsilon polypeptides are replaced with TFP.

In fig. 2 is a schematic illustration showing examples of reprogrammed T-cell receptor fusion polypeptide (TFP) variants of the present invention. The illustration, designated scFv:TCR-Va, shows an example of a reprogrammed TCR containing TFP that contains an anti-mesothelin scFv and a full-length TCR-Va polypeptide fused through a (G ₄ S) ₃ linker sequence. The illustration, designated scFv:TCRVa:TCR-Ve, shows an example of a reprogrammed TCR that contains multiple TFPs, including i) an anti-mesothelin scFv and a full-length TCR-Va polypeptide fused via a (G ₄ S) ₃ linker sequence, and ii) an anti-mesothelin scFv and mesothelin and the full-length TCR-Ve polypeptide fused via a (G ₄ S) ₃ linker sequence. The illustration, designated scFv:ΔTCRVa:CD3ε, shows an example of a reprogrammed TCR that contains multiple TFPs, including i) an anti-mesothelin scFv and a truncated (Δ) TCR polypeptide fused via a (G ₄ S) ₃ linker sequence, and ii) an anti-mesothelin scFv and a full-length CD3 epsilon polypeptide fused via a (G ₄ S) ₃ linker sequence. The truncated (Δ) TCR polypeptide is truncated by Va deletion. The illustration, designated scFv:ATCR-Va:ΔTCR-Vβ, shows an example of a reprogrammed TCR that contains multiple TFPs, including i) an anti-mesothelin scFv and a truncated (Δ) TCR Va polypeptide fused through a (G ₄ S) ₃ linker sequence, and ii) anti-mesothelin scFv and a truncated (Δ) TCR Ve polypeptide fused via a (G ₄ S) ₃ linker sequence. The truncated (Δ) TCR polypeptide is truncated by Ve deletion.

In fig. 3 is a schematic illustration showing the use of T cell receptor fusion polypeptides (TFPs) of the present invention. An example TFP contains an anti-mesothelin VH domain and a full-length CD3 epsilon polypeptide fused via a (G ₄ S) ₃ linker sequence. When TFP is produced by a T cell or introduced into a T cell, it binds to other endogenous T cell receptor (TCR) polypeptides (shown as including two CD3 epsilon polypeptides, one CD3 gamma polypeptide, one CD3 delta polypeptide, two CD3 zeta polypeptide, one TCR alpha subunit and one TCR beta subunit, with the horizontal gray segment representing the plasma membrane) to form a reprogrammed TCR in which one or both endogenous CD3 epsilon polypeptides are replaced with TFP.

In fig. 4 is a series of schematic illustrations showing DNA constructs encoding various TFPs.

In fig. 5A depicts an example of TFP surface expression assay on activated PBMCs and shows CD3 ⁺ cells (APC vs CD3 gate) activated with MSLN TFP and stained for CD8 (APCy7 vs CD8 y-axis) and mesothelin (MSLN) (Rphycoerythrin-labeled hMSLN IgG Zenon®, x-axis). Shown from left to right are cells that were either not transduced or transduced with anti-MSLN-CD3ε TFP, anti-MSLN-CD28ζ CAR, and anti-MSLN-41BBζ CAR constructs.

In fig. 5B depicts an example of a TFP surface expression assay on activated PBMCs and shows cells activated with self-single domain TFPs stained for MSLN Fc and assayed for ZPB. Top row (left to right) shows untransduced cells as well as cells transduced with control anti-MSLN-CD3ε TFP

- 6 043737 (SSI). Rows 2-4 show binding antibodies against MSLN SD1, SD4 and SD6, respectively, in cells transduced with (from left to right) CD3ε TFP, CD3yTFP, TCRe TFP and CD28Z constructs

ZFB-tagged CARs.

In fig. 6A is an example graph demonstrating the killing of mesothelin (MSLN) positive target HeLa cells (adenocarcinoma of the cervix, ATCC® CCL-2™) by anti-MSLN-TFP constructs over time. Activated PBMCs were untreated (trace 1), untransduced (trace 2), or transduced with empty vector (trace 3), with anti-MSLNCD3ε TFP (trace 4), anti-MSLN-CD28ζ CAR, or anti-MSLN-41BBζ CAR and expanded in for 8 days before incubation with 1x10 ⁴ MSLN-positive HeLa target cells.

In fig. 6B is an example graph showing the killing of MSLN-negative HeLa target cells (adenocarcinoma of the cervix, ATCC® CCL-2™) by anti-MSLNTFP constructs over time. Activated PBMCs were untreated (trace 1), untransduced (trace 2), or transduced with empty vector (trace 3), with anti-MSLN-CD3ε TFP (trace 4), anti-MSLN-CD28ζ CAR, or anti-MSLN-41BBζ CAR, and propagated for 8 days before incubation with 1x104 MSLN-positive HeLa target cells.

In fig. Figure 6C shows the killing of MSLN-positive cells in high MSLN expressing cell lines (HeLa cells) using T cells obtained from two different human donors (top and bottom). Cell killing tracks are shown for TFP-T cells with self-binding anti-MSLN antibodies SD1 (Figure 7A), SD4 (middle), and SD6 (right). Activated PBMCs were untransduced (trace 1) or transduced with CD3ε TFP (trace 2), CD3y TFP (trace 3), TCRe TFP (trace 4), or CD28Z CAR. The normalized cell index, indicating cytotoxicity, was established using a real time cell analyzer (RTCA) assay.

In fig. 7 shows a series of graphs demonstrating the binding activity of CAR-T cells against MSLN and TFP-T cells against a target cell line expressing high levels of mesothelin (HeLa-Luc( ^{MSLN hlgh} )). Shown is the % of cells killed in samples in the absence of T cells (target cells only), with empty vector (NT) transduced T cells, anti-MSLN T cells (positive control), or anti-mesothelin TFP-T cells with self binding antibodies against mesothelin SD1 (Figure 7A), SD4 (Figure 7B) and SD6 (Figure 7C), each in the format CD3ε TFP, CD3y TFP, TCRe TFP and CD28Z CAR. In each graph, black bars represent a 1:1 ratio of T cells to target cells and gray bars represent a 1:5 ratio of T cells to target cells. Similar results were observed for the second T-cell donor.

In fig. 8 shows a series of graphs demonstrating anti-MSLN CAR and TFP-T cell activity against a target cell line expressing low levels of mesothelin (PC3-MSLN( /low)). Shown is the % of cells killed in samples in the absence of T cells (target cells only), empty vector transduced (NT), anti-MSLN (positive control, SS1), or native anti-mesothelin constructs SD1, SD4, and SD6 in TFP CD3ε formats (Figure 8A), CD3y (Figure 8B), TCRe (Figure 8C) and CAR CD28Z (Figure 8D). In each graph, black bars represent a 1:1 ratio of T cells to target cells and gray bars indicate a 1:5 ratio of T cells to target cells. Similar results were observed for the second T-cell donor.

In fig. 9 shows the results of FACS analysis demonstrating the activation of T cells expressing CAR and TFP constructs against MSLN when co-cultured with MSLN+ cells. As shown in FIG. 9A, from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cells cocultured with MSLN− cells are shown in the top row, and cells cocultured with MSLN+ target cells are shown in the bottom row. The cells were then stained with antibodies specific to the surface activation markers CD69 and CD25 or the cytolytic granule component granzyme B (GrB). The number of cells stained with anti-CD69 antibodies corresponds to the x-axis, and the number of cells stained with anti-CD25 antibodies corresponds to the y-axis. As shown, T cells expressing anti-mesothelin CAR and TFP constructs were activated by culture with MSLN+ cells, as indicated by increased expression levels of CD69 and CD25, relative to coculture with MSLN− cells (Fig. 9B). The percentage of CD25+ cells for each construct in MSLN− (white bars) and MSLN+ (black bars) cells is shown. A similar experiment was performed using K562 MSLN cells (circles) and K562 MSLN+ cells (squares) in either untransduced T cells or T cells transduced with a positive control anti-MSLN binding antibody (510-SS1-CD3ε) (Figure 9C). Data represent the sum of CD25+, CD69+, and CD25+/CD69+ cells. In fig. 9D shows data for self-binding anti-MSLN antibodies SD1 (squares), SD4 (circles), and SD6 (triangles) in MSLN-targeted K562 cells (left panel) and MSLN+ K562 cells (right panel) combined with donor T cells. having TFP formats CD3ε, CD3y, TCRe and

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CD28Z CAR. Similar results were observed using cells from a second T cell donor.

In fig. 10 shows the results of FACS analysis demonstrating the activation of T cells expressing CAR and TFP constructs against MSLN when co-cultured with MSLN+ cells. Cells were stained for surface antigens with anti-CD3 APC (gate) and anti-CD8 APCy7 (y-axis) before fixation, permeabilization, and staining with Alexafluor700 against granzyme B (x-axis). As shown in FIG. 10A, from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cells cocultured with MSLN− cells are shown in the top row, and cells cocultured with MSLN+ target cells are shown in the bottom row. CD8 T cells expressing CAR and TFP constructs against mesothelin were activated by culture with MSLN+ cells, as shown by increased levels of intracellular GrB, relative to coculture with MSLN− cells (Fig. 10B). The percentage of granzyme B (GrB+) cells for each construct when cocultured with MSLN− (white bars) or MSLN+ (black bars) cells is shown. In fig. 11 shows the results of an ELISA analysis of cytokine production in activated T cells expressing CAR and TFP constructs against MSLN when co-cultured with K562 cells overexpressing MSLN. K562 cells overexpressing BCMA were used as negative controls. After 24 hours, cells were analyzed for expression of IFN-γ (Fig. 11A) and IL-2 (Fig. 11B) by ELISA. As shown in each FIG. from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN28ζ CAR, or anti-MSLN-41BBζ CAR. Cells cocultured with MSLN − cells are indicated by white bars, and cells cocultured with MSLN+ target cells are indicated by black bars.

In fig. 12 shows a series of graphs demonstrating the in vivo efficacy of a TFP-T cell MSLN-specific sdAb in a murine xenograft model of mesothelioma. Mice were inoculated with luciferase-labeled MSTO-211H-FL-MSLN-Luc at 1 x 106 cells per mouse, tumors were grown until tumor volume was approximately 300 mm ³ , and each animal was injected intravenously with 1 x 10 ⁷ T cells. In fig. 12A shows the tumor volume after injection of T cells, including from left to right control - no T cells, SD1 CD3ε-TFP, and SD4 CD3ε-TFP. In fig. 12B shows CD3y-TFP with binding antibodies SD1 and SD4, as well as SD1 CD28Z CAR. In fig. 12C-D show the results of surviving mice from 12A-B that were reinjected with tumor cells to determine whether the mice would maintain their immunity to MSLN without a second injection of T cells. Mice that had previously been tumor-free with SD1 CD3ε TFP T cells (12C) and SD1 CD3y TFP T cells (12D) were reinoculated with MSLN+ (MSTO) or MSLN− (Raji) tumor cell lines. Tumor volume was measured and plotted on the x-axis.

In fig. 13 shows the generation and functional analysis of MSLN-TFP-T cells obtained from ovarian cancer patients. In fig. 13A is a schematic diagram of the experimental design. In fig. 13B-C shows the in vitro destruction of MSTO-MSLN-Luc SD1 tumor cells by ε-TFP-T cells from the patient. MSTO-MSLN-Luc tumor cells (target cells) were confirmed to express mesothelin (13B); SD1 ε-TFP T cells (effector cells) and the corresponding non-transduced control were added at ratios of effector cells to target cells of 5 to 1, 1 to 1, or 1 to 5 for 24 hours. The luminescence of target cells was measured in relative luminescence units (RLU) using an M5 SpectraMax® plate reader (Molecular devices). Each line in the figure represents the average of 3 replicates (13C). In fig. 13D-L shows measurement of the cytokine profile of SD1 ε-TFP-T cells obtained from patients with ovarian cancer, including IFNy (13D), GM-CSF (13E), granzyme A (13F), granzyme B (13G), IL- 2 (13H), MIP-1a (13I), MIP-1e (13J), TNFa (13K) and perforin (13L). MSTOMSLN-Luc tumor cells (target cells) were plated at a concentration of 10,000 cells/well in a flat-bottomed 96-well plate. SD1 ε-TFP-T cells (effector cells) and the corresponding non-transduced control were added at a ratio of 1 to 1 for 24 hours. Cell supernatants were collected and cytokines were measured using the Luminex® assay. In fig. 14 shows the in vivo efficacy of patient-derived SD1 CD3ε-TFP-T cells in a high-MSLN murine xenograft tumor model. MSTO-211H-FL MSLN-Luc cells were inoculated subcutaneously at 1x106 cells per mouse. Ten days after tumor injection (tumor volume ~200-300 mm ³ ), each animal was intravenously injected with 5x106 T cells. Each line on the figure represents one animal. Data are shown for T cells from ND12 (14A), patient 1 (14B), patient 2 (14C), patient 3 (14D), and patient 4 (14E). Circles indicate tumor size in mice inoculated with untransduced T cells; squares indicate tumor size in mice inoculated with TFP-T cells.

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Detailed Description of the Invention

In one aspect, disclosed herein are isolated nucleic acid molecules encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit and a human or humanized antibody domain containing an anti-mesothelin binding domain. In some embodiments, the TCR subunit comprises an extracellular TCR domain. In other embodiments, the TCR subunit comprises a TCR transmembrane domain. In yet other embodiments, the TCR subunit comprises a TCR intracellular domain. In further embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit . In other further embodiments, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from a CD3 epsilon, CD3 gamma or CD3 delta intracellular signaling domain, or an amino acid sequence further having at least one, two or three modifications. In other further embodiments, the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional 4-1BB signaling domain and/or a functional CD3 zeta signaling domain, or an amino acid sequence further having at least one, two or three modifications.

In some embodiments, the isolated nucleic acid molecules comprise (i) light chain (LC) CDR1, LC CDR2, and LC CDR3 of any amino acid sequence of the anti-mesothelin light chain binding domain provided herein, and/or (ii) heavy chain (HC) CDR1 ), HC CDR2 and HC CDR3 of any amino acid sequence of the anti-mesothelin heavy chain binding domain provided herein.

In some embodiments, the light chain variable region comprises an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications of the light chain variable region amino acid sequence provided herein, or a sequence from 95-99 % identity with the amino acid sequence presented herein. In other embodiments, the heavy chain variable region comprises an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications of the heavy chain variable region amino acid sequence provided herein, or a sequence from 95-99 % identity with the amino acid sequence presented herein. In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of T cell receptor alpha or beta chain, CD3 delta, CD3 epsilon, or CD3 gamma, or functional fragments, or an amino acid sequence, which also has at least one, two or three modifications, but not more than 20, 10 or 5 modifications. In other embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha, beta chain, or TCR subunits CD3 epsilon, CD3 gamma, or CD3 delta, or a functional fragment thereof, or an amino acid a sequence that also has at least one, two or three modifications, but not more than 20, 10 or 5 modifications. In some embodiments, the encoded TFP includes a transmembrane domain that contains a transmembrane domain of a protein selected from the group consisting of a TCR alpha, beta, or zeta chain or CD3 epsilon, CD3 gamma, and CD3 delta CD45, CD2, CD4, CD5 , CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a functional fragment thereof, or an amino acid sequence further having at least one, two or three modifications , but no more than 20, 10 or 5 modifications. In some embodiments, the encoded anti-mesothelin binding domain is connected to the TCR extracellular domain via a linker sequence. In some cases, the encoded linker sequence contains (G ₄ S) _n , where n = 1 to 4. In some cases, the encoded linker sequence contains a long linker (L) sequence. In some cases, the encoded long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the encoded linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence encoded contains (G ₄ S) _n , where n = 1 to 3.

In some embodiments, the isolated nucleic acid molecules further comprise a sequence encoding a co-stimulatory domain. In some cases, the co-stimulatory domain is a functional signaling domain derived from a protein selected from the group consisting of DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD 18), ICOS (CD278) and 4-1BB (CD137), or an amino acid sequence also having at least one, two or three modifications, but not more than 20, 10 or 5 modifications.

In some embodiments, the isolated nucleic acid molecules further comprise a leader sequence.

Also provided herein are isolated polypeptide molecules encoded by any of the previously described nucleic acid molecules.

Also provided herein in another aspect are isolated T cell receptor fusion protein (TFP) molecules that comprise a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the isolated TFP molecules comprise an antibody or antibody fragment comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.

In some embodiments, the domain of a human or humanized antibody comprises an antibody fragment. In some embodiments, the domain of a human or humanized antibody comprises a scFv or a _VH domain.

In some embodiments, the anti-mesothelin binding domain is an scFv or a VH domain. In other embodiments, the anti-mesothelin binding domain comprises a light chain and a heavy chain of the amino acid sequence provided herein, or a functional fragment thereof, or an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications to the amino acid sequence of the light chain variable region provided herein, or a sequence with 95-99% identity to the amino acid sequence provided herein.

In some embodiments, the isolated TFP molecules comprise an extracellular domain of a TCR that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a T cell receptor alpha or beta chain, CD3 delta, CD3 epsilon or CD3 gamma, or an amino acid sequence , which also has at least one, two or three modifications, but not more than 20, 10 or 5 modifications.

In some embodiments, the anti-mesothelin binding domain is connected to the TCR extracellular domain via a linker sequence. In some cases, the linker region contains (G ₄ S) _n , where n = 1 to 4. In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence comprises (G ₄ S) _n , where n = 1 to 3. In some embodiments, the isolated TFP molecules further comprise a sequence encoding a co-stimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.

Also provided herein are vectors that contain a nucleic acid molecule encoding any of the previously described TFP molecules. In some embodiments, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, the nucleotide sequence in the vector further comprises a poly(A) tail. In some embodiments, the nucleic acid sequence in the vector further comprises a 3'UTR.

Also provided herein are cells that contain any of the described vectors. In some embodiments, the cell is a human T cell. In some embodiments, the cell is a CD8+ or CD4+ T cell. In other embodiments, the cells further comprise a nucleic acid encoding an inhibitory molecule that contains a first polypeptide that contains at least a portion of the inhibitory molecule linked to a second polypeptide that contains a positive signal from an intracellular signaling domain. In some cases, the inhibitory molecule comprises a first polypeptide that contains at least a portion of PD1, and a second polypeptide that contains a co-stimulatory domain and a primary signaling domain.

In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-mesothelin binding domain, an extracellular TCR domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.

In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-mesothelin binding domain, an extracellular TCR domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functional integration into the endogenous TCR complex. In another aspect, the present document provides human CD8+ or CD4+ T cells that contain at least two TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, on, and/or on the surface of a human CD8+ or CD4+ T cell.

In another aspect, provided herein are protein complexes that comprise i) a TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex.

In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of T cell receptor alpha or beta chain, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-mesothelin binding domain is connected to the extracellular domain of the TCR via a linker sequence. In some cases, the linker region contains (G ₄ S) _n , where n = 1 to 4. In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence contains (G ₄ S) _n , where n = 1 to 3.

Also provided herein are human CD8+ or CD4+ T cells that contain at least two different TFP proteins per protein complex described.

In another aspect, provided herein is a population of human CD8+ or CD4+ T cells, wherein the T cell population, individually or collectively, comprises at least two TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functional interaction with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, on and/or on the surface of a human CD8+ or CD4+ T cell.

In another aspect, provided herein is a population of human CD8+ or CD4+ T cells, wherein the T cells of the population, individually or collectively, contain at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein.

In another aspect, provided herein are methods for generating a cell, comprising transducing a T cell with any of the described vectors.

In another aspect, provided herein are methods for producing a population of RNA engineered cells, comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA contains a nucleic acid encoding any of the described TFP molecules.

In another aspect, provided herein are methods of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell expressing any of the disclosed TFP molecules. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.

In another aspect, provided herein are methods of treating a mammal with a disease associated with mesothelin expression, comprising administering to the mammal an effective amount of a cell containing any of the disclosed TFP molecules. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease such as cancer or malignancy, or a precancerous condition such as pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, or endometrial cancer, or is non-cancerous similar indication associated with mesothelin expression.

In some embodiments, cells expressing any of the described TFP molecules are administered in combination with a substance that alleviates one or more side effects associated with administration of a cell expressing the TFP molecule. In some embodiments, cells expressing any of the described TFP molecules are administered in combination with an agent that treats a disease associated with mesothelin.

Also provided herein for use as a drug are any of the described isolated nucleic acid molecules, any of the described isolated polypeptide molecules, any of the described isolated TFPs, any of the described protein complexes, any of the described vectors or any of the described cells.

Definitions.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention relates.

The singular form is used to denote one or more than one (i.e. at least one) grammatical object. By way of example, an element means one element or more than one element.

As used herein, the term about can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 25, 30, or more than 30 percent, which will depend on the situation and will be known or understood by one skilled in the art.

As used herein, the subject, or subjects, or individuals may include, but are not limited to, mammals, such as humans or non-human mammals, such as domesticated, farmed or wild animals, as well as birds and aquatic animals. Patients are subjects suffering from, at risk of developing, or otherwise in need of a disease, disorder or condition, or otherwise in need of the compositions and methods provided herein. As used herein, the terms treat or treatment refer to any sign of success in treating or alleviating a disease or condition. Treatment may include, for example, reducing, slowing, or alleviating the severity of one or more symptoms of a disease or condition, or it may include reducing the frequency of symptoms of a disease, defect, disorder, undesirable condition, or the like in a patient. As used herein, the term treat or prevent is sometimes used to refer to a method that results in a certain level of treatment or alleviation of a disease or condition and involves a number of results aimed at achieving this goal, including, but not limited to, complete prevention of the condition. As used herein, the term prevention refers to preventing a disease or condition, such as tumor formation, in a patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present invention and does not subsequently develop a tumor or other form of cancer, then the disease in that individual will be prevented for at least a certain period of time. period of time.

As used herein, a therapeutically effective amount is that amount of a composition or an active component thereof that is sufficient to provide a beneficial effect or otherwise reduce the detrimental effect on the individual to whom the composition is administered. The term "therapeutically effective dose" as used herein means a dose that produces one or more of the desired or preferred (eg, beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dosage will depend on the purpose of treatment and will be determined by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

As used herein, a T cell receptor (TCR) fusion protein or TFP includes a recombinant polypeptide derived from various TCR-containing polypeptides that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, usually when colocated in the T cell or on its surface. A TFP-T cell is a T cell that has been transduced (eg, in accordance with the methods described herein) and that expresses TFP, eg, included in a natural TCR. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+/CD8+ T cell. In some embodiments, the TFP-T cell is an NK cell. In some embodiments, the TFP-T cell is a gamma delta T cell. As used herein, the term mesothelin, also known as MSLN, or CAK1 antigen, or pre-pro-megakaryocyte potentiating factor, refers to the protein that is encoded by the MSLN gene (or megakaryocyte potentiating factor (MPF)) in humans. Mesothelin is a 40 kDa protein that is present on normal mesothelial cells and is overexpressed in several human tumors, including mesothelioma and adenocarcinoma of the ovary and pancreas. The mesothelin gene encodes a precursor protein that is processed to produce mesothelin, which is attached to the cell membrane via a glycophosphatidylinositol linkage and a soluble 31-kDa moiety called megakaryocyte potentiating factor (MPF). Mesothelin may be involved in cell adhesion, but its biological function is unknown. Mesothelin is a tumor differentiation antigen that is typically present on mesothelial cells lining the pleura, peritoneal cavity, and pericardium. Mesothelin is an antigenic determinant found on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cells and some squamous cell carcinomas (see, for example, Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. Cancer Res. 12:42254231(2006)). Mesothelin interacts with CA125/MUC16 (see, for example, Rump et al., J. Biol. Chem.

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279:9190-9198(2004) and Ma et al., J. Biol. Chem. 287:33123–33131(2012)). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human mesothelin can be found under UniProt/Swiss-Prot accession number Q13421. The canonical polypeptide sequence of human mesothelin can be found under UniProt accession number Q13421 (or Q13421-1): MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLCLAHRLSEPPEDL DALPLDLLLFLNPDAFSGPQACTRFFSRITKANVD LLPRGAPERQRLLPAALACWGVRGSLLSEADVRA LGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLL PVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWE LEACVDAALLATQMDRVNAIPFTYEQLDVLKH KLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVT SLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSS VPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSM DLATFMKLRTDAVLPLTVAEVQK LLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGY LVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO: 15). The nucleotide sequence encoding human mesothelin transcript variant 1 can be found under accession number NM005823. The nucleotide sequence encoding human mesothelin transcript variant 2 can be found under accession number NM013404. The nucleotide sequence encoding human mesothelin transcript variant 3 can be found under accession number NM001177355. Mesothelin is expressed on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cells and squamous cell carcinomas (see, for example, Kojima et al., J. Biol. Chem. 270:21984-21990 (1995) and Onda et al., Clin. Cancer Res 12:4225–4231(2006)). Other cells expressing mesothelin are presented below in the definition of mesothelin expression disease. Mesothelin also interacts with CA125/MUC16 (see, for example, Rump et al., J. Biol. Chem. 279:9190-9198 (2004) and Ma et al., J. Biol. Chem. 287:33123-33131 ( 2012)). In one example, the antigen binding portion of TFP recognizes and binds to an epitope within the extracellular domain of the mesothelin protein that is expressed on a normal or malignant mesothelioma cell, ovarian cancer cell, pancreatic adenocarcinoma cell, or squamous cell carcinoma cell.

As used herein, the term antibody refers to protein or polypeptide sequences derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies may be intact immunoglobulins of polyclonal or monoclonal origin or fragments thereof and may be obtained from natural or recombinant sources.

The terms antibody fragment or antibody binding domain refer to at least one antibody moiety or recombinant variants thereof that contain an antigen binding domain, i.e., an antigen defining variable region of an intact antibody that will be sufficient to provide recognition and specific binding of the antibody fragment to the target, such as an antigen and its specific epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments, single chain (sc) Fv antibody fragments (scFv), linear antibodies, single domain antibodies (abbreviated sdAb) ( V _L or VH), camel VHH domains and multispecific antibodies formed from antibody fragments.

The term scFv refers to a fusion protein containing at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are continuously linked by a short flexible polypeptide linker and capable of being expressed as a single polypeptide chain, with the scFv retaining the specificity of the intact antibody from which it was derived. A heavy chain variable region or _VH (or in the case of single domain antibodies such as nanobodies, _VHH ) in relation to an antibody refers to a heavy chain fragment that contains three CDRs interspersed with flanking regions known as framework regions, these framework regions being known as generally more highly conserved than CDRs and form a scaffold to support the CDRs.

As used herein, unless otherwise noted, a scFv may have the V _L and V _H variable regions in any order, for example, relative to the N-terminal or C-terminal ends of the polypeptide, the scFv may contain a VL linker-V _H or V _H -linker-VL. The portion of the TFP composition of the present invention containing the antibody or fragment thereof may exist in various forms where the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb) or a heavy chain antibody HCAb, single chain antibody ( scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual , Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of the TFP composition of the present invention comprises an antibody fragment. In an additional aspect, the TFP comprises an antibody fragment that contains a scFv or sdAb.

The term antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which generally defines the class to which the antibody belongs.

The term antibody light chain refers to the smaller of two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa () and lambda () light chains refer to the two main isotypes of antibody light chains. The term recombinant antibody refers to an antibody that is produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody that has been produced by synthesizing a DNA molecule encoding an antibody and which expresses an antibody protein, or an amino acid sequence defining an antibody, wherein the DNA or amino acid sequence has been produced using recombinant DNA technology or an amino acid sequence that is available and well known in the art.

The term antigen or Ag refers to a molecule that is capable of being specifically bound by an antibody or that otherwise provokes an immune response. Such an immune response may involve either the production of antibodies, or the activation of specific immunocompetent cells, or both.

One skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Moreover, antigens can be obtained from recombinant or genomic DNA. One skilled in the art will appreciate that any DNA that contains a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response thereby encodes an antigen as that term is used herein. Moreover, one skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be appreciated that the present invention includes, but is not limited to, the use of portions of the nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, one skilled in the art will appreciate that an antigen does not necessarily have to be encoded by a gene. It will be appreciated that the antigen may be formed by synthesis, or may be obtained from a biological sample, or may be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell or fluid with other biological components.

The term antitumor effect refers to a biological effect that can manifest itself in a variety of ways, including, but not limited to, reducing tumor volume, reducing the number of tumor cells, reducing the number of metastases, increasing life expectancy, reducing tumor cell proliferation, reducing tumor cell survival, or facilitating various physiological symptoms associated with the cancerous condition. The antitumor effect may also be reflected in the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent a tumor from occurring in the first place.

The term autologous refers to any material obtained from the same individual to whom it will subsequently be administered.

The term allogeneic refers to any material obtained from another animal of the same species or from a different patient than the individual to whom the material will be administered. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material obtained from individuals of the same species may be genetically different enough to cause antigenic interaction.

The term xenogeneic refers to a graft obtained from an animal of another species.

The term cancer refers to a disease that is characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer , lung cancer, etc. The phrase mesothelin expression disease includes, but is not limited to, a disease associated with mesothelin expression or a condition associated with cells that express mesothelin, including, for example, proliferative diseases such as cancer , or a malignant tumor, or a precancerous condition. In one aspect, the cancer is mesothelioma. In one aspect, the cancer is pancreatic cancer. In one aspect, the cancer is ovarian cancer. In one aspect, the cancer is gastric cancer. In one aspect, the cancer is lung cancer. In one aspect, the cancer is endometrial cancer. Non-cancer related indications associated with mesothelin expression include, but are not limited to, autoimmune disease (eg, lupus, rheumatoid arthritis, colitis), inflammatory diseases (allergy and asthma), and transplantation.

The term conservative modifications in sequences refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be made to the antibody or antibody fragment of the present invention using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine , phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the TFP of the present invention can be replaced with other amino acid residues from the same side chain family, and the altered TFP can be tested using the functional assays described herein.

The term stimulation refers to the primary response induced by the binding of a stimulatory domain or stimulatory molecule (eg, a TCR/CD3 complex) to its recognition ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules and/or reorganization of cytoskeletal structures, etc.

The term stimulatory molecule or stimulatory domain refers to a molecule or portion thereof expressed by a T cell that provides primary cytoplasmic signaling sequences that regulate the primary activation of the TCR complex by the stimulatory pathway for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated through, for example, the binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, resulting in the mediation of a T cell response including, but not limited to, proliferation, activation, differentiation, etc. Primary Cytoplasmic Signaling Sequence (also called a primary signaling domain) that acts in a stimulatory pathway may contain a signaling motif that is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences for specific use in the present invention include, but are not limited to, TCR-derived zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as ICOS) and CD66d.

The term antigen presenting cell or APC refers to a cell of the immune system, such as a support cell (eg, B cell, dendritic cell, etc.), that displays a foreign major histocompatibility complex (MHC) antigen on its surface. T cells can recognize such complexes using T cell receptors (TCRs). APCs process antigens and present them to T cells. As used herein, the term intracellular signaling domain refers to the intracellular portion of the molecule. The intracellular signaling domain generates a signal that induces immune effector function in a TFP-containing cell, such as a TFP-expressing T cell. Examples of immune effector function, for example, in a TFP-expressing T cell include cytolytic and helper T cell activity, including cytokine secretion. In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include domains derived from molecules that are responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include domains derived from molecules that are responsible for costimulatory signals or antigen-dependent stimulation.

The primary intracellular signaling domain may contain an ITAM (immunoreceptor tyrosine activating motif). Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, CD3 derivatives zeta, FcR gamma, FcR

- 15 043737 beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d DAP10 and DAP12.

The term costimulatory molecule refers to a recognizable binding partner on a T Cell that specifically binds to a costimulatory ligand, thereby mediating a T cell costimulatory response such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecule, BTLA and Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 ( CD11a/CD 18) and 4-1BB (CD137). The costimulatory intracellular signaling domain may be an intracellular portion of a costimulatory molecule. The co-stimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C , SLAMF7, NKp80, CD160, B7-H3 and a ligand that specifically binds to CD83, etc. The intracellular signaling domain may comprise all or all of the native intracellular signaling domain of the molecule from which it was derived, or a functional fragment thereof. The term 4-1BB refers to a member of the TNFR superfamily with the amino acid sequence provided under GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mice, rodents, marmosets, apes, etc.; and the costimulatory domain 4-1BB is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues in non-human species, e.g., mice, rodents, marmosets, apes, etc.

The term coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a specific sequence of amino acids, as well as the biological properties resulting from this. Thus, a gene, cDNA, or RNA encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and usually provided in sequence listings, and the non-coding strand used as a template for transcription of a gene or cDNA can be considered to encode a protein or other product of that gene or cDNA.

Unless otherwise indicated, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or RNA may also include introns, to the extent that a nucleotide sequence that encodes a protein may, in some versions, contain one or more introns.

The terms effective amount or therapeutically effective amount are used interchangeably herein and refer to the amount of a compound, formulation, material or composition as described herein that is effective to achieve a particular biological or therapeutic result.

The term endogenous refers to any material derived from or produced within an organism, cell, tissue, or system.

The term exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term expression refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.

The term transfer vector refers to a composition of material that contains an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, ionic or amphiphilic linked polynucleotides, plasmids, and viruses. Thus, the term transfer vector includes a plasmid or virus capable of autonomous replication. The term should also be taken to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

The term expression vector refers to a vector containing a recombinant polynucleotide that contains expression control sequences operably linked to the nucleotide sequence to be expressed.

- 16 043737

The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that contain a recombinant polynucleotide.

The term lentivirus refers to a genus in the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect nondividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vector options. HIV, simian immunodeficiency virus, feline immunodeficiency virus are examples of lentiviruses.

The term lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, including in particular a self-inactivating lentiviral vector as proposed by Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical practice include, but are not limited to, Oxford BioMedica's LENTFVECTOR™ gene delivery technology, Lentigen's LENTIMAX™ vector system, etc. Non-clinical types of lentiviral vectors are also available and known. a person skilled in the art. The term homologous or identity refers to the identity of a subunit sequence between two polymer molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit position in both molecules is occupied by the same monomeric subunit, for example, if the position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is directly proportional to the number of matching or homologous positions; for example, if half (eg, five positions in a ten-subunit-long polymer) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (eg 9 out of 10) match or are homologous, the two sequences are 90% homologous.

Humanized forms of antibodies of non-human origin (for example, murine) are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding antibody sequences) that contain a minimal sequence derived from immunoglobulin of non-human origin. In most cases, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which the complementarity determining region (CDR) residues of the recipient are replaced with CDR residues of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and potency. In some examples, the Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues of non-human origin. Moreover, the humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications can further improve and optimize the effectiveness of the antibodies or antibody fragments. In general, a humanized antibody or antibody fragment will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to such non-human immunoglobulin regions, and all or a substantial portion of the FR regions are immunoglobulin sequence regions person. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically from human immunoglobulin. For more information, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

Human or fully human refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule will be of human origin or will consist of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

The term isolated means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living being is not isolated, but the same nucleic acid or peptide, partially or completely separated from coexisting materials in its natural state, will be isolated. The isolated nucleic acid or protein may exist in a substantially purified form or may exist in a foreign environment, such as, for example, a host cell. In the context of the present invention, the following abbreviations are used for commonly occurring nucleic acid bases. A is for adenosine, C is for cytosine, G is for guanosine, T is for thymidine, and U is for uridine. The term operably linked or transcriptional control refers to the functional relationship between a regulatory sequence and a heterologous nucleotide sequence

- 17 043737 activity, which leads to the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is operably linked to a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. Functionally linked DNA sequences may be adjacent to each other and, for example, if it is necessary to connect two protein-coding regions, be in the same reading frame.

The term parenteral administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM) or intrasternal injection, intratumoral administration or infusion methods.

The term nucleic acid or polynucleotide refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in single- or double-stranded form. Unless specifically limited, the term covers nucleic acids that contain known analogues of naturally occurring nucleotides, have binding properties similar to those of the reference nucleic acid, and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs (single nucleotide polymorphisms), and complementary sequences, as well as explicitly stated sequence. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more of the selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms peptide, polypeptide and protein are used interchangeably and refer to a compound containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and is not limited by the maximum number of amino acids that the protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, which in the art are also often called peptides, oligopeptides and oligomers, for example, and longer chains, which in the art are usually called proteins, of which there are many types. . Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes a naturally occurring peptide, a recombinant peptide, or a combination thereof.

The term promoter refers to a DNA sequence recognized by the cellular transcription machinery or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

The term promoter/regulatory sequence refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are necessary for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

The term constitutive promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell.

The term inducible promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes production of the gene product in a cell essentially only when an inducer that matches that promoter is present in the cell.

The term tissue-specific promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene or gene-determinant, causes production of a gene product in a cell essentially only if the cell is a cell of the tissue type that corresponds to the promoter.

The terms linker and flexible polypeptide linker, as used in the context of scFv, refer to a peptide linker that consists of amino acids, such as glycine and/or serine residues, used alone or in combination to link together a heavy chain variable region and a light chain variable region. In one embodiment, the flexible polypeptide linker

- 18 043737 is a Gly/Ser linker and contains the amino acid sequence (Gly-Gly-Gly-Ser) _n , where n is a positive integer that is equal to or greater than 1. For example, n=1, n=2, n= 3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly ₄ Ser) ₄ or (Gly ₄ Ser) ₃ . In another embodiment, the linkers include multiple (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser) repeats. Also included within the scope of the present invention are the linkers described in WO2012/138475 (incorporated herein by reference). In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence contains (G4S)n, where n = 1 to 3.

As used herein, a 5' cap (also called RNA cap, 7methylguanosine RNA cap, or m7G RNA cap) is a modified guanine nucleotide that is added to the front or 5-end of eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group that is linked to the first transcribed nucleotide. Its presence is critical for ribosome recognition and protection against RNases. Cap addition is transcriptionally coupled and occurs cotranscriptionally in such a way that one influences the other. Shortly after the start of transcription, the 5-end of the synthesized mRNA binds to the cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required to cap the mRNA. Synthesis proceeds as a multi-stage biochemical reaction. The capping moiety can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

As used herein, the term in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro. In general, in vitro transcribed RNA is obtained from an in vitro transcription vector. An in vitro transcription vector contains a template that is used to produce in vitro transcribed RNA.

As used herein, poly(A) is a series of adenosines attached by polyadenylation to mRNA. In a preferred embodiment of the transient expression construct, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences may be modified chemically or enzymatically to modulate the functionality of the mRNA, such as localization, stability or translation efficiency.

As used herein, the term polyadenylation refers to the covalent bonding of a polyadenyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3'po1y(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a polyadenylation signal is added to transcripts that contain a specific sequence, a polyadenylation signal. The Po1y(A) tail and its associated protein help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but may additionally occur later in the cytoplasm. After transcription ends, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. A cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After mRNA cleavage, adenosine residues are added to the free 3' end at the cleavage site.

As used herein, transient refers to the expression of a non-integrated transgene for a period of time, expressed in hours, days or weeks, where the period of time of expression is less than the period of time for expression of a gene integrated into the genome or contained in a stable plasmid replicon in a cell - owner. The term signal transduction pathway refers to the biochemical relationship between various signaling molecules that play a role in transmitting a signal from one part of the cell to another part of the cell. The phrase cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting it across the cell membrane.

The term subject is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

The term essentially purified cell refers to a cell that is substantially free of other cell types. An essentially purified cell also refers to a cell that has been separated from the other types of cells with which it is normally associated in its natural state. In some cases, the population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, these cells are cultured in vitro. In other aspects, these cells are not cultured in vitro.

As used herein, the term therapeutic means treatment. The therapeutic effect is achieved by reducing, suppressing, remitting or eliminating a disease state.

As used herein, the term prophylaxis means the prevention of a disease or disease state or its prophylactic treatment. In the context of the present invention, a tumor antigen or a hyperproliferative disease antigen or an antigen associated with a hyperproliferative disease refers to antigens that are characteristic of specific hyperproliferative diseases. In some aspects, the hyperproliferative disease antigens of the present invention were derived from cancers, including, but not limited to, primary or metastatic melanoma, mesothelioma, renal cell carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer colon, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney, endometrial and stomach cancer. In some cases, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian cancer, mixed ovarian Müllerian carcinoma, endometrioid mucinous ovarian carcinoma, pleural malignancy, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, serous carcinoma uterus, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, mammary adenocarcinoma, mesothelin expression disease and combinations thereof, mesothelin expression disease and combinations thereof.

The term transfected or transformed or transduced refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A transfected, transformed, or transduced cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. A cell includes the subject's primary cell and its progeny.

The term specifically binds refers to an antibody, antibody fragment, or specific ligand that recognizes and binds to a recognizable binding partner (eg, mesothelin) present in a sample, but which will not necessarily or substantially recognize and bind to other molecules in that sample.

Ranges: Throughout this specification, various aspects of the invention may be represented by ranges. It should be understood that the description in terms of ranges is provided for convenience and brevity only and should not be construed as inflexibly limiting the scope of the present invention. Accordingly, the range description should be considered to specifically disclose all possible subranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 up to 6, etc.; as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something either 95%, 96%, 97%, 98% or 99% identical, and also includes subranges such as 96-99%, 96-98%, 9697%, 97-99%, 97-98% and 98-99% identical. This applies regardless of the band width.

Description

Provided herein are compositions of matter and methods of use for treating a disease such as cancer using T cell receptor (TCR) fusion proteins. As used herein, a T cell receptor (TCR) fusion protein or TFP includes a recombinant polypeptide derived from various TCR-containing polypeptides that is generally capable of i) binding to a surface antigen on target cells and ii) interact with other polypeptide components of the intact TCR complex, usually when colocated in the T cell or on its surface. As proposed herein, TFPs provide significant advantages over chimeric antigen receptors. The term chimeric antigen receptor or alternatively CAR refers to a recombinant polypeptide containing an extracellular antigen binding domain in the form of a scFv, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as intracellular signaling domains) containing a functional signaling domain derived from a stimulatory molecule, as defined below. In general, the central intracellular signaling domain of CAR is derived from the CD3 zeta chain, which is typically associated with the TCR complex. The CD3 zeta signaling domain may be fused to one or more functional signaling domains derived from at least one co-stimulatory molecule, such as 4-1BB (ie, CD137), CD27 and/or CD28.

T cell receptor (TCR) fusion proteins (TFP).

The present invention provides recombinant DNA constructs encoding TFP, wherein the TFP comprises an antibody fragment that specifically binds to mesothelin, e.g., human mesothelin, wherein the sequence of the antibody fragment is adjacent to a nucleic acid sequence encoding a TCR subunit or a portion thereof, and is in the same reading frame with it. The TFPs provided herein are capable of binding to one or more endogenous (or alternatively, one or more exogenous or a combination of endogenous and exogenous) TCR subunits to form a functional TCR complex.

In one aspect, the TFP of the present invention contains a target-specific binding element, otherwise known as an antigen binding domain. The choice of fragment depends on the type and number of target antigens that define the surface of the target cell. For example, the antigen binding domain may be selected to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease condition. Thus, examples of cell surface markers that can act as target antigens for the antigen binding domain in the TFP of the present invention include markers associated with viral, bacterial and parasitic infections; autoimmune diseases and cancers (eg malignant diseases).

In one aspect, a TFP-mediated T cell response can be directed to an antigen of interest by inserting an antigen binding domain into TFP that specifically binds to the desired antigen.

In one aspect, the antigen binding domain portion of the TFP comprises an antigen binding domain that targets mesothelin. In one aspect, the antigen binding domain targets human mesothelin.

An antigen binding domain can be any domain that binds an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and functional fragments thereof, including, but not limited to, a single domain antibody such as a severe antibody variable domain. chain (VH), variable light chain domain (V _L ) and variable domain (VHH) nanobodies of camel origin, as well as with an alternative scaffold known in the art that functions as an antigen binding domain, such as that of recombinant fibronectin, anticalin, DARPIN, etc. Likewise, a natural or synthetic ligand that specifically recognizes and binds to a target antigen can be used as the antigen binding domain for TFP. In some cases, it would be useful for the antigen binding domain to be derived from the same species in which the TFP will ultimately be used. For example, for use in humans, it would be useful for the antigen binding domain of TFP to contain human or humanized residues for the antigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) of light chain complementarity determining region 1 (CDR1 LC), complementarity determining region 2 light chain (CDR2 LC), and complementarity determining region 3 light chain (CDR3 LC) of the humanized or human anti-mesothelin binding domain described herein, and/or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (CDR1 HC), the complementarity determining region 2 of the heavy chain (CDR2 HC ) and the complementarity determining region 3 heavy chain (CDR3 HC) of the humanized or human anti-mesothelin binding domain described herein, e.g., the humanized or human anti-mesothelin binding domain comprises one or more, e.g., all three, CDR LC and one or more eg all three, CDRHC. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) of a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, for example, a humanized or human anti-mesothelin binding domain has two heavy chain variable regions, each containing an HC CDR1, HC CDR2 and HC CDR3 described herein . In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized or human light chain variable region as described herein and/or a humanized or human heavy chain variable region as described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized heavy chain variable region as described herein, for example, at least two humanized or human heavy chain variable regions as described herein. In one embodiment, the anti-mesothelin binding domain is a scFv comprising a light chain and a heavy chain of the amino acid sequence provided herein. In one embodiment, the binding domain vs.

- 21 043737 mesothelin (for example, scFv) contains: a light chain variable region containing an amino acid sequence having at least one, two or three modifications (for example, substitutions), but not more than 30, 20 or 10 modifications (for example, substitutions) a light chain variable region amino acid sequence provided herein, or a sequence with 95-99% identity to the amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of the heavy chain variable region amino acid sequence represented by herein, or a sequence with 95-99% identity to the amino acid sequence presented herein. In one embodiment, the humanized or human anti-mesothelin binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein is attached to a heavy chain variable region containing an amino acid sequence described herein via a linker, e.g. , a linker described herein. In one embodiment, the humanized anti-mesothelin binding domain includes a linker (Gly ₄ -Ser) _n , where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4. Light chain variable region and heavy chain variable region scFvs can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region. In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence contains (G4S)n, where n = 1 to 3.

In some aspects, the non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody that is naturally produced in a human, or a fragment thereof. In one aspect, the antigen binding domain is humanized.

The humanized antibody can be produced using various techniques known in the art, including, but not limited to, CDR grafting (see, for example, European Patent No. EP 239,400; International Publication No. WO 91/09967 and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089, the entire contents of each of which are incorporated herein by reference), masking of surface residues or surface modification (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/ 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814 and Roguska et al., 1994, PNAS, 91:969-973, each of which is included herein in its entirety by reference), circuit permutation (see, for example, US Patent No. 5,565,332, the entire contents of which are incorporated herein by reference), as well as techniques described, for example, in US Patent Application Publication No. US2005/0042664, US Patent Application Publication No. US2005/0042664, US Patent No. US2005/0048617, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng. , 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp) :5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994) and Pedersen et al. , J. Mol. Biol., 235(3):959-73 (1994), the entire contents of each of which are incorporated herein by reference. Often, framework residues in framework regions will be replaced by a corresponding residue from the donor antibody CDR to alter, for example, improve binding to the antigen. These framework substitutions are identified using methods well known in the art, for example, by modeling interactions of CDRs and framework residues to identify framework residues important for antigen binding, and sequence comparisons to identify unusual framework residues at specific positions (see, for example, Queen et al., US Pat. No. 5,585,089 and Riechmann et al., 1988, Nature, 332:323, the entire contents of each of which are incorporated herein by reference).

A humanized antibody or antibody fragment has one or more amino acid residues remaining therein from a non-human source. Such amino acid residues of non-human origin are often referred to as imported residues, which are typically taken from an imported variable domain. As provided herein, humanized antibodies or antibody fragments contain one or more CDRs of immunoglobulin molecules and framework regions of non-human origin, wherein the amino acid residues containing the framework are derived entirely or substantially from the human germ line. Various techniques for humanizing antibodies or antibody fragments are well known in the art and can advantageously be carried out following the method of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323 -327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by

- 22 043737 replacement of CDRs or CDR sequences of rodents with the corresponding sequences of a human antibody, i.e., CDR grafting (EP 239,400; PCT publication No. WO 91/09967 and US patent No. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference). In such humanized antibodies and antibody fragments, the substantially less-than-intact human variable domain has been replaced by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some of the CDR residues, and possibly some of the framework (FR) residues, are replaced with residues from the analogous regions of rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by masking surface residues, or altering the surface (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7 (6):805-814 (1994) and Roguska et al., PNAS, 91:969-973 (1994)), or strand permutation (US Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

The choice of human variable domains, both light and heavy, to be used to create humanized antibodies is to reduce antigenicity. The so-called best-fit method screens the variable domain sequence of a rodent antibody against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then taken as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol. , 196:901 (1987), the entire contents of which are incorporated herein by reference. Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subset of light or heavy chains. A similar framework can be used for several different humanized antibodies (see eg Nicholson et al Mol Immun 34 (16-17): 1157-1165 (1997) Carter et al Proc Natl Acad Sci USA 89:4285 (1992) Presta et al ., J. Immunol., 151:2623 (1993), the entire contents of which are incorporated herein by reference.) In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region is derived from the germline sequence VH4- 4-59. In one embodiment, the framework region may contain one, two, three, four or five modifications, such as substitutions, for example, from an amino acid in the corresponding mouse sequence. In one embodiment, the framework region, for example, all four light chain variable region frameworks are derived from the VK3-1.25 germline sequence. In one embodiment, the framework region may contain one, two, three, four or five modifications, such as substitutions, for example, from an amino acid in the corresponding mouse sequence.

In some aspects, the portion of the TFP composition of the present invention that contains the antibody fragment is humanized while maintaining high affinity for the target antigen and other advantageous biological properties. In accordance with one aspect of the invention, humanized antibodies and antibody fragments are obtained by analyzing parent sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are publicly available and known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected potentially useful immunoglobulin sequences. Examination of such mappings allows analysis of the likely role of residues in the functioning of potential immunoglobulin sequences, for example, analysis of residues that affect the ability of a candidate immunoglobulin to bind a target antigen. Therefore, FR residues can be selected and combined from recipient and imported sequences such that a characteristic of the desired antibody or antibody fragment, such as increased affinity for a target antigen, is achieved. In general, CDR residues are directly and significantly involved in influencing antigen binding.

The humanized antibody or antibody fragment may retain similar antigenic specificity as the parent antibody, for example, in the present invention, the ability to bind to human mesothelin. In some embodiments, a humanized antibody or antibody fragment may have improved binding affinity and/or specificity for human mesothelin.

In one aspect, the anti-mesothelin binding domain is characterized by particular functional features or properties of the antibody or antibody fragment. For example, in one aspect, the portion of the TFP composition of the present invention that contains the antigen binding domain specifically binds to human mesothelin. In one aspect, the antigen binding domain has the same or similar binding specificity to human mesothelin as the FMC63 scFv described by Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention provides an antigen binding domain comprising an antibody or antibody fragment, wherein the antigen binding domain specifically binds to the protein mesothelin or a fragment thereof, wherein

- 23 043737 an antibody or antibody fragment contains a variable light chain and/or a variable heavy chain that includes the amino acid sequence presented herein. In some aspects, the scFv is adjacent to and in reading frame with the leader sequence.

In one aspect, the anti-mesothelin binding domain is a fragment, for example, a single chain variable fragment (scFv). In one aspect, the anti-mesothelin binding domain is an Fv, Fab, (Fab')2, or a bifunctional (eg, bispecific) fusion antibody (eg, Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof described herein bind to the protein mesothelin with wild-type or increased affinity. Also provided herein are methods of producing an antigen binding domain of an antibody specific for a target antigen (e.g., mesothelin or any target antigen described elsewhere herein to target the binding domains of a fusion fragment), the method including providing: addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a _VH domain provided herein, wherein a _VH domain that is a variant amino acid sequence of a _VH domain optionally combines the VH domain so provided with one or more VL domains and testing the VH domain or _VH / _VL combination or combinations to identify a particular binding member or antigen binding domain of an antibody that is specific for a target antigen of interest (eg, mesothelin) and optionally has one or more desired properties. In some cases, the _VH and scFv domains can be prepared in accordance with a method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc Natl Acad Sci USA 85:5879-5883). ScFv molecules can be produced by linking together the VH and VL regions using flexible polypeptide linkers. scFv molecules contain a linker (eg, a Ser-Gly linker) with an optimized length and/or amino acid composition. The length of the linker can greatly influence how the variable regions fold and interact. In fact, if a short polypeptide linker is used (eg between 5 and 10 amino acids), intrachain folding is prevented. Intrastrand folding is also required to bring two variable regions together to form a functional epitope binding site. In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G ₄ S) _n , where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence contains (G ₄ S) _n , where n = 1 to 3. For examples of linker orientation and dimensions, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, US Patent No. 7,695,936, US Patent Application Publications Nos. 20050100543 and 20050175606, as well as in PCT publications Nos. WO2006/020258 and WO2007/024715, all of which are incorporated herein by reference.

scFv may contain a linker of about 10, 11, 12, 13, 14, 15 or more than 15 residues between its V _L and V _H regions. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and series. In another embodiment, the linker sequence contains sets of glycine and serine repeats such as (Gly ₄ Ser) _n , where n is a positive integer that is equal to or greater than 1. In one embodiment, the linker may be (Gly ₄ Ser)4 or (Gly ₄ Ser) ₃ . Variations in linker length can maintain or enhance activity, resulting in superior performance in activity studies. In some cases, the linker sequence contains a long linker (L) sequence. In some cases, the long linker sequence contains (G4S)n, where n = 2 to 4. In some cases, the linker sequence contains a short linker (CL) sequence. In some cases, the short linker sequence contains (G ₄ S) _n , where n = 1 to 3.

Stability and mutations.

The stability of the anti-mesothelin binding domain of, for example, scFv molecules (eg, soluble scFv) can be assessed relative to the biophysical properties (eg, thermal stability) of a conventional control scFv molecule or full-length antibody. In one embodiment, the humanized or human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7, 5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius compared to the parent scFv in the described assays . The improved thermal stability of the anti-mesothelin binding domain, such as scFv, will subsequently be inherent in the entire mesothelin-TFP construct, leading to improved therapeutic properties of the anti-mesothelin TFP construct. The thermal stability of the binding domain against mesothelin, e.g. scFv, can be improved by at least

- 24 043737 at least about 2°C or 3°C compared to a traditional antibody. In one embodiment, the anti-mesothelin binding domain, eg scFv, has 1°C improved thermal stability compared to a conventional antibody. In another embodiment, the anti-mesothelin binding domain, eg scFv, has 2°C improved thermal stability compared to a conventional antibody. In another embodiment, the scFv is improved by 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14° C or 15°C thermal stability compared to traditional antibody. Comparisons can be made, for example, between the scFv molecules described herein and the scFv molecules or Fab fragments of the antibody from which the VH and _VL scFvs were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM may be measured. Methods for measuring TM and other methods for determining protein stability are described below. Mutations in scFv (resulting from humanization or mutagenesis of soluble scFv) alter scFv stability and improve the overall stability of scFv and the anti-mesothelin TFP construct. The stability of the humanized scFv is compared with respect to the mouse scFv using measurements such as T _M , temperature denaturation and temperature aggregation. In one embodiment, the anti-mesothelin binding domain, eg, scFv, contains at least one mutation resulting from the humanization process such that the mutated scFv provides improved stability in the anti-mesothelin TFP construct. In another embodiment, the anti-mesothelin binding domain, e.g., scFv, contains at least 1.2, 3.4, 5, 6, 7, 8, 9, 10 mutations resulting from the humanization process such that the mutated scFv provides improved stability in the mesothelin-TFP construct.

In one aspect, the antigen binding domain of TFP contains an amino acid sequence that is homologous to the amino acid sequence of the antigen binding domain described herein, and the antigen binding domain retains the desired functional properties of the anti-mesothelin antibody fragments described herein. In one particular aspect, the TFP composition of the present invention contains an antibody fragment. In a further aspect, the antibody fragment comprises scFv. In various aspects, the TFP antigen binding domain is constructed by modifying one or more amino acids within one or both variable regions (e.g., VH and/or _VL ), for example, within one or more CDR regions and/or within one or more framework regions . In one particular aspect, the TFP composition of the present invention contains an antibody fragment. In a further aspect, the antibody fragment comprises scFv.

One skilled in the art will appreciate that an antibody or antibody fragment of the present invention may be further modified such that it differs in amino acid sequence (eg, from wild type), but not in the desired activity. For example, additional nucleotide substitutions resulting in amino acid substitutions at non-essential amino acid residues can be performed on a protein. For example, a nonessential amino acid residue in a molecule may be replaced by another amino acid residue from the same side chain family. In another embodiment, the amino acid strand may be replaced by a structurally similar strand that differs in the order and/or composition of the side chain family members, for example, a conservative substitution may be made in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

Percent identity, in the context of two or more nucleic acid sequences or polypeptide sequences, refers to two or more sequences that are the same. Two sequences are substantially identical if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76% , 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% identity in the specified region or, if not specified, in the entire sequence), when compared or aligned for maximum agreement in the comparison window or designated region, as determined using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of

- 25 043737 length from 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids). For sequence comparisons, typically one sequence serves as a reference sequence to which the sequences of interest are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the sequence are indicated and the software parameters of the algorithm for working with sequences are indicated. You can use the default program settings or specify alternative settings. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences compared to the reference sequence based on software parameters. Methods for aligning sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by a local homology algorithm according to Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, homologous alignment algorithm according to Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, similarity search according to Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA) or manual alignment and visual inspection (see ., for example, Brent et al., (2003) Current Protocols in Molecular Biology). Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. In one aspect, the present invention provides modifications to the original amino acid sequence of an antibody or fragment (eg, scFv) that form functionally equivalent molecules. For example, the VH or _VL binding domain against mesothelin, for example, scFv contained in TFP, can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the original framework region of the V _H or V _L binding domain against mesothelin, eg scFv. The present invention provides for modifications to the entire TFP construct, eg, modifications to one or more amino acid sequences of different domains of the TFP construct, to provide functionally equivalent molecules. The TFP design can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the original TFP construct.

Extracellular domain.

The extracellular domain can be obtained from either a natural or recombinant source. If the source is natural, the domain can be derived from any protein, but in particular from a membrane-associated or transmembrane protein. In one aspect, the extracellular domain is capable of binding to the transmembrane domain. The extracellular domain for particular use in the present invention may include at least extracellular regions, for example, T cell receptor alpha, beta or zeta chains or CD3 epsilon, CD3 gamma or CD3 delta, or in alternative embodiments CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

Transmembrane domain.

In general, the TFP sequence contains an extracellular domain and a transmembrane domain, encoded by a single genomic sequence. In alternative embodiments, TFP may be designed to include a transmembrane domain that is heterologous to the extracellular domain of TFP. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein was derived (e.g., 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, or up to 15 amino acids of the extracellular region), and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein was derived (e.g., ., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region). In one aspect, a transmembrane domain is used that is associated with one of the domains in TFP. In some cases, the transmembrane domain may be selected from or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of similar or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another TFP on the surface of a TFP-T cell. In another aspect, the amino acid sequence of the transmembrane domain can be modified or substituted so as to minimize interactions with the binding domains of a native binding partner that is present in the same TFP.

- 26 043737

The transmembrane domain can be obtained from either a natural or recombinant source. If the source is natural, the domain can be formed from any membrane-associated or transmembrane protein. In one aspect, the transmembrane domain is capable of transmitting signals to intracellular domains whenever TFP has bound a target. The transmembrane domain for particular use in the present invention may include at least transmembrane regions, for example, T cell receptor alpha, beta or zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some cases, the transmembrane domain may be attached to the extracellular region of TFP, such as the antigen binding domain of TFP, via a hinge, such as a human protein hinge. For example, in one embodiment, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge or a CD8a hinge. Linkers

Optionally, a short oligo- or polypeptide linker of 2 to 10 amino acids in length may form a link between the transmembrane domain and the cytoplasmic region of the TFP. The glycineserine doublet provides a particularly suitable linker. For example, in one aspect, the linker contains the amino acid sequence GGGGSGGGGS (SEQ ID NO. 53). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO. 54).

Cytoplasmic domain.

The cytoplasmic domain of TFP may include an intracellular signaling domain if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha and TCR beta subunits generally do not contain a signaling domain. The intracellular signaling domain is typically responsible for activating at least one of the normal effector functions of the immune cell into which TFP has been introduced. The term effector function refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term intracellular signaling domain refers to the part of the protein that transmits the signal to effector function and directs the cell to perform a specialized function. While typically the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact circuit as long as it will signal the effector function. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to signal effector function.

Examples of intracellular signaling domains for use in the TFP of the present invention include cytoplasmic T cell receptor (TCR) sequences and coreceptors that act in concert to initiate signal transduction upon engagement of antigen receptors, as well as any derivative or variant of these sequences and any recombinant sequence with the same functional ability.

It is known that TCR-generated signals alone are not sufficient to fully activate intact T cells and that a secondary and/or co-stimulatory signal is required. Thus, it can be said that activation of naïve T cells can be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCRs (primary intracellular signaling domains), and those that act in an antigen-independent manner to mediate secondary or costimulatory signal (secondary cytoplasmic domain, e.g. costimulatory domain).

The primary signaling domain regulates the primary activation of the TCR complex either through a stimulatory pathway or an inhibitory pathway. Primary intracellular signaling domains that act through stimulatory pathways may contain signaling motifs that are known as immunoreceptor tyrosine activating motifs (ITAMs).

Examples of ITAMs containing primary intracellular signaling domains that are intended for specific use in the present invention include ITAMs CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In one embodiment, the TFP of the present invention contains an intracellular signaling domain, for example, a CD3 epsilon primary signaling domain. In one embodiment, the primary signaling domain comprises a modified ITAM domain, eg, a mutated ITAM domain that has changed (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified primary intracellular signaling domain containing an ITAM, for example, an optimized and/or truncated primary intracellular signaling domain containing an ITAM. In one embodiment, the primary signaling domain contains one, two, three, four or more ITAM motifs. The intracellular signaling domain of TFP may contain a CD3 zeta signaling domain.

- 27 043737 but or in combination with any other desired intracellular signaling domains suitable in the context of the TFP of the present invention. For example, the intracellular signaling domain of TFP may contain part of the CD3 epsilon chain and a co-stimulatory signaling domain. The costimulatory signaling domain refers to the portion of TFP containing the intracellular domain of the costimulatory molecule. A costimulatory molecule is a cell surface molecule, distinct from an antigen receptor or its ligands, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, DAP10, DAP12, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C , B7-H3 and a ligand that specifically binds to CD83, etc. For example, costimulation of CD27 has been shown to enhance the expansion, effector function and survival of human TFP-T cells in vitro and increase the persistence and antitumor activity of human T-cells. cells in vivo (Song et al. Blood. 2012; 119(3):696-706).

The intracellular signal sequences in the cytoplasmic portion of the TFP of the present invention may be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form a link between intracellular signal sequences.

In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, can be used as a suitable linker.

In one aspect, a TFP-expressing cell described herein may further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., for the same target (mesothelin) or for a different target (e.g., CD123) . In one embodiment, when a TFP-expressing cell contains two or more different TFPs, the antigen binding domains of the different TFPs may be those that will not interact with each other. For example, a cell expressing a first and a second TFP may have the antigen binding domain of the first TFP, for example, as a fragment, for example, a scFv, which will not bind to the antigen binding domain of the second TFP, for example, the antigen binding domain of the second TFP is V _HH . In another aspect, the TFP-expressing cell described herein may further express another substance, for example, a substance that enhances the activity of the TFP-expressing cell. For example, in one embodiment, the substance may be one that inhibits an inhibitory molecule. Inhibitory molecules, such as PD1, may in some embodiments reduce the ability of a TFP-expressing cell to elicit an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, the inhibitory molecule inhibitor substance comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to a cell, such as an intracellular signaling domain described herein. In one embodiment, the substance contains a first polypeptide, for example, of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of them (for example, at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (for example, containing a costimulatory domain (for example, 4-1BB, CD27 or CD28, for example, as described herein) and/or a primary signaling domain (e.g., CD3 zeta signaling domain described herein.) In one embodiment, the substance comprises a first PD1 polypeptide or fragment thereof (e.g., at least a portion of a PD1 extracellular domain) and a second intracellular signaling domain polypeptide described herein (e.g. , CD28 signaling domain, as described herein, and/or CD3 zeta signaling domain, as described herein.) PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been demonstrated to reduce T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261 -8;Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is common in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094 ). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the substance comprises an extracellular domain (ECD) of an inhibitory molecule, for example, programmed cell death protein 1 (PD-1) may be fused to a transmembrane domain and optionally an intracellular signaling domain, such as 41BB and CD3 zeta (also referred to herein as PD1 TFP). In one embodiment, PD1 TFP, when used in combination with the anti-mesothelin TFP described herein, improves T cell persistence. In one embodiment, the TFP is a PD1 TFP containing the extracellular domain of PD 1. Alternatively, TFPs are provided comprising an antibody or antibody fragment, such as scFv, that specifically binds programmed cell death ligand 1 (PD-L1) or programmed cell death ligand 2 (PD-L2).

In another aspect, the present invention provides a population of TFP-expressing T cells, for example, TFP-T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, a population of TFP-T cells may include a first cell expressing a TFP having an anti-mesothelin binding domain described herein, and a second cell expressing a TFP having a different anti-mesothelin binding domain, e.g. an anti-mesothelin domain described herein, which is different from the anti-mesothelin binding domain in the TFP expressed by the first cell. As another example, a population of TFP-expressing cells may include a first cell expressing a TFP that includes an anti-mesothelin binding domain, for example, as described herein, and a second cell expressing a TFP that includes an antigen binding domain for a target other than mesothelin (eg, another tumor-associated antigen).

In another aspect, the present invention provides a population of cells, wherein at least one cell in the population expresses TFP having an anti-mesothelin domain described herein, and the second cell expresses another substance, for example, a substance that increases the activity of TFP-expressing cells. For example, in one embodiment, the substance may be one that inhibits an inhibitory molecule. Inhibitory molecules, for example, can in some embodiments reduce the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PDL2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, the inhibitory molecule inhibitor substance comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to a cell, such as an intracellular signaling domain described herein.

Described herein are methods for producing in vitro transcribed RNA encoding TFP. The present invention also includes an RNA coding construct for TFP that can be directly transfected into a cell. A method for producing mRNA for use in transfection may involve in vitro transcription (IVT) of a template using specially designed primers followed by the addition of polyA to produce a construct containing 3' and 5' untranslated sequence (UTR), 5' cap and/or an internal ribosome entry site (IRES), a nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. The RNA obtained in this way can effectively transfect various types of cells. In one aspect, the matrix includes sequences for TFP.

In one aspect, TFP against mesothelin is encoded by messenger RNA (mRNA). In one aspect, mRNA encoding TFP against mesothelin is introduced into a T cell to produce a TFP-T cell. In one embodiment, in vitro transcribed TFP RNA can be introduced into a cell as a form of transient transfection. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro synthesis of mRNA using appropriate primers and RNA polymerase. The source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA sequence of synthetic DNA, or any other suitable DNA source. The desired template for in vitro transcription is the TFP of the present invention. In one embodiment, the DNA for use in PCR contains an open reading frame. DNA can be derived from a naturally occurring DNA sequence from an organism's genome. In one embodiment, the nucleic acid may include some or all of the 5' and/or 3' untranslated regions (UTR). Nucleic acid may include exons and introns. In one embodiment, the DNA for use in PCR is a human nucleic acid sequence. In another embodiment, the DNA for use in PCR is a human nucleic acid sequence, including 5' and 3' UTRs. The DNA may alternatively be an artificial DNA sequence that is not typically expressed in naturally occurring organisms. An example of an artificial DNA sequence is one that contains a portion of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together may come from a single organism or from more than one organism.

PCR is used to create a template for in vitro transcription of mRNA that is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to contain regions substantially complementary to the regions of DNA that will be used as a template for PCR. As used herein, essentially complementary refers to nucleotide sequences in which most or all of the bases in the primer sequence are complementary, or one or more bases are not complementary or mismatched. Substantially complementary sequences are capable of renaturation or hybridization with the intended DNA target under the renaturation conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify the portion of nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to enhance the portion of the nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to enhance the coding region of the human cDNA, including all or part of the 5' and 3' UTRs. Primers used in PCR can be prepared by synthesis methods that are well known in the art. Forward primers are primers that contain a stretch of nucleotides that are substantially complementary to nucleotides on the DNA template that are located upstream in the DNA sequence that is to be amplified. Upstream is used herein to designate the location 5, in the DNA sequence that is to be amplified, relative to the coding strand. Reverse primers are primers that contain a region of nucleotides that are substantially complementary to the double-stranded DNA template that are located downstream in the DNA sequence that is to be amplified. Below is used herein to indicate the location 3' in the DNA sequence that is to be amplified relative to the coding strand.

Any DNA polymerase used in PCR can be used in the methods described herein. The reagents and polymerase are commercially available from various sources. Chemical structures capable of providing translation stability and/or efficiency may also be used. RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between one and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences that will be added to the coding region can be changed in various ways, including, but not limited to, designing PCR primers that anneal to different UTR regions. Using this approach, one skilled in the art will be able to modify the length of the 5' and 3' UTRs necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be natural, endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by introducing UTR sequences into forward and reverse primers or through any other modifications to the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest may be useful in modifying the stability and/or translation efficiency of RNA. For example, it is known that AU-rich elements in 3'UTR sequences can reduce mRNA stability. Thus, 3' UTRs can be selected or designed to enhance the stability of the transcribed RNA based on properties of the UTRs that are well known in the art.

In one embodiment, the 5' UTR may comprise a Kozak sequence of an endogenous nucleic acid. Alternatively, by adding a 5' UTR that is not endogenous to the nucleic acid of interest by PCR as described above, the Kozak consensus sequence can be altered by adding the 5' UTR sequence. The Kozak sequence can improve the translation efficiency of some RNA transcripts, but it does not appear to be required for all RNAs to ensure efficient translation. The need for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR may be the 5' UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to prevent degradation of the mRNA by exonucleases.

To allow RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter should be attached to the DNA template upstream of the transcribed sequence. When a sequence that acts as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes included in the PCR product upstream of the transcribed open reading frame. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

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In a preferred embodiment, the mRNA contains a cap at the 5-end and a 3-po1y(A) tail, which determine ribosome binding, translation initiation and stability of the mRNA in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemer product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR produces normal-sized mRNA, which is not efficient for eukaryotic transfection, even if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The traditional way to integrate polyA/T regions into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can lead to plasmid instability, which is the reason why plasmid DNA templates obtained from bacterial cells are often heavily contaminated with deletions and other aberrations. This makes cloning procedures not only labor-intensive and time-consuming, but often unreliable. Therefore, a method that allows the construction of DNA templates with a polyA/T 3' region without the use of cloning is highly desirable.

The PolyA/T segment of the DNA transcription template can be obtained by PCR using a reverse primer containing a polyT tail, such as a 100 T tail (size can be 50-5000 T), or after PCR by any other method, including, among other things, DNA ligation or in vitro recombination. Po1y(A) tails also ensure RNA stability and reduce their degradation. In general, the length of the pol·y(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is from 100 to 5000 adenosines.

Po1y(A) RNA tails can be further extended after in vitro transcription using a poly(A) polymerase such as E. Coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the pol·y(A) tail from 100 nucleotides to 300 to 400 nucleotides results in an approximately twofold increase in RNA translation efficiency. In addition, the addition of various chemical groups to the 3' end can increase the stability of the mRNA. Such attachment may contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogues can be incorporated into the po1y(A) tail by pol·y(A) polymerase. ATP analogues can further increase RNA stability. 5' caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced using the methods described herein include a 5' cap. The 5' cap is prepared using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958 -966 (2005)).

RNAs produced using the methods described herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or man-made sequence that initiates cap-independent binding of the ribosome to the mRNA and promotes translation initiation. Any cell electroporation solutes may be added, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants and surfactants.

RNA can be introduced into target cells using any of a number of different methods, for example commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 ( BTX) (Harvard Instruments, Boston, MA) or Gene Pulser II electroporator (BioRad, Denver, CO), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as gene guns (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

Nucleic acid constructs encoding TFP/

The present invention also provides nucleic acid molecules encoding one or more of the TFP constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is represented as a DNA construct.

Nucleic acid sequences targeting the desired molecules can be obtained by recombinant methods known in the art, such as by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to contain the gene, or by direct isolation from cells and tissues containing it using standard techniques. Alternatively, the gene of interest may be obtained synthetically rather than by cloning.

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The present invention also provides vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentivirus, are useful tools for achieving long-term gene transfer, as they ensure long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the added benefit of low immunogenicity.

In another embodiment, the vector containing the nucleic acid encoding the desired TFP of the present invention is an adenoviral vector (A5/35). In another embodiment, expression of nucleic acids encoding TFP can be accomplished using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nuclease (see June et al. 2009 Nature Reviews Immunol. 9.10: 704-716, included this document by reference).

The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art (see, for example, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference). In another embodiment, the invention provides a vector for gene therapy.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector, including, but not limited to, a plasmid, phagemid, phage derivative, animal virus, and cosmid. Vectors of particular interest include expression vectors, replication vectors, probe acquisition vectors, and sequencing vectors.

Moreover, the expression vector can be delivered into the cell in the form of a viral vector. Viral vector technologies are well known in the art and are described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular science references. biology. Viruses used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease recognition sites, and one or more selectable markers (for example, WO 01/96584; WO 01/29058 and US Pat. No. 6,326,193) .

A number of virus-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. As a rule, they are located in the region at 30-110 bp. upstream of the start site, although a number of promoters have also been demonstrated to contain functional elements downstream of the start site. Often the spacing between promoter elements is flexible so that promoter function is maintained when elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function either together or independently to activate transcription.

An example of a promoter capable of expressing a TFP transgene in a mammalian T cell is the EF1a promoter. The native EF1a promoter stimulates the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA into the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in stimulating TFP expression from transgenes cloned into a lentiviral vector (see, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)). Another example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, murine mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter , avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, actin promoter, myosin promoter, elongation factor-1 promoter, hemoglobin promoter

- 32 043737 bin and creatine kinase promoter. Moreover, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch capable of driving expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or stopping expression when it is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter. To assess the expression of a TFP polypeptide or portions thereof, the expression vector introduced into a cell may also contain a selectable marker gene, a reporter gene, or both to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected with viral agents. vectors. In other aspects, the selectable marker may be carried by a single region of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to allow expression in the host cell. Suitable selectable markers include, for example, antibiotic resistance genes such as neo, etc.

Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue and that encodes a polypeptide whose expression results in some easily detectable property, such as enzyme activity. Expression of the reporter gene is analyzed at an appropriate time after introduction of DNA into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene (eg, Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or purchased commercially. In general, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to assess the ability of substances to modulate promoter-activated transcription.

Methods for introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 14, Cold Spring Harbor Press, NY). One method of introducing a polynucleotide into a host cell is transfection with calcium phosphate.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for introducing genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentivirus, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc. (see, for example, US Patent Nos. 5,350,674 and 5,585,362.

Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An example of a colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). Other prior art methods for targeted delivery of nucleic acids are available, such as nanoparticle targeted polynucleotide delivery or other suitable submicron element delivery system.

In the case of a non-viral delivery system, an example of a delivery vehicle is a liposome. The use of lipid formulations to introduce nucleic acids into a host cell (in vitro, ex vivo or in vivo) is contemplated. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed throughout the lipid bilayer of the liposome, attached to the liposome through a linker molecule that is associated with both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, may be suspended in a lipid, contained in or associated with

- 33 043737 complexed with a micelle or otherwise associated with a lipid. Compositions associated with a lipid, lipid/DNA or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, like micelles, or have a disrupted structure. They may also simply be dispersed in solution, presumably forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Suitable lipids can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (DMPC) is available from Sigma, St. Louis, MO; Dicetyl phosphate (DCP) is available from K & K Laboratories (Plainview, NY); cholesterol (Choi) is available from Calbiochem-Behring; Dimyristylphosphatidylglycerol (DMPG) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Lipid stock solutions in chloroform or chloroform/methanol can be stored at approximately -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. Liposome is a general term covering various single-layer and multilayer lipid carriers obtained through the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as containing vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes contain many lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement to form closed structures and trap water and solutes between lipid bilayers (Ghosh et al., 1991, Glycobiology 5: 505-10). However, compositions that have structures in solution other than the normal vesicular structure are also covered. For example, lipids can adopt a micellar structure or exist only in the form of heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also provided. Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to the inhibitor of the present invention, a number of assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, molecular biology assays well known to those skilled in the art, such as Southern and Northern blotting, RTPCR and PCR; biochemical assays, such as detecting the presence or absence of a specific peptide, for example, by immunological methods (ELISA and Western blots) or by the assays described herein, to identify substances within the scope of the present invention.

The present invention further provides a vector containing a nucleic acid molecule encoding TFP. In one aspect, the TFP vector can be directly transduced into a cell, such as a T cell. In one aspect, the vector is a cloning or expression vector, for example, a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, dual microchromosomes), retroviral and lentiviral vectors. In one aspect, the vector is capable of expressing a TFP construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

Sources of T cells/

Before expansion and genetic modification, a source of T cells is obtained from the subject. The term subject is intended to include living organisms in which an immune response can be elicited (eg, mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art can be used. In some aspects of the present invention, T cells can be obtained from a unit of blood collected from a subject using a variety of techniques known to one skilled in the art, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained through apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for further processing steps. In one aspect of the present invention, the cells are washed with phosphate-buffered saline (PBS). In an alternative aspect, the rinse solution does not contain

- 34 043737 calcium and may not contain magnesium or may not contain many, if not all, divalent cations. The initial stages of activation in the absence of calcium can lead to increased activation. As those skilled in the art will appreciate, the washing step can be accomplished by methods known to those skilled in the art, such as using a semi-automated flow centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions . After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be directly resuspended in the culture medium.

In one aspect, T cells are isolated from peripheral blood lymphocytes by erythrocyte lysis and monocyte depletion, for example, by PERCOLL™ gradient centrifugation or countercurrent centrifugal elutriation. A specific subset of T cells, such as CD3 ⁺ , CD28 + , CD4 ⁺ , CD8 ⁺ , CD45RA + and CD45RO + T cells, can be further isolated through positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/CD28 antibody-conjugated beads (e.g., 3x28), such as DYNABEADS™ M-450 CD3/CD28 T cells, for a period of time sufficient to positively select the desired T cells. -cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period is in the range of 30 minutes to 36 hours or more for all integer values in this range. In a further aspect, the time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred aspect, the time period is from 10 to 24 hours. In one aspect, the incubation time is 24 hours. Longer incubation times can be used to isolate T cells in any situation where there are fewer T cells relative to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Moreover, using longer incubation times may improve the efficiency of CD8+ T cell capture. Thus, by simply decreasing or increasing the time at which T cells bind to CD3/CD28 beads and/or increasing or decreasing the ratio of beads to T cells (as described later herein), positive or negative selection of T cell subsets can be preferentially performed early on. cultivation or at other times during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, positive or negative selection of T cell subsets can be advantageously performed at the start of culture or at other desired times. One skilled in the art will appreciate that multiple selection steps can also be used in the context of the present invention. In certain aspects, it may be desirable to perform a selection procedure and use unselected cells in the activation and expansion process. Unselected cells can also be subjected to additional selection steps. Enrichment of the T cell population through negative selection can be achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells through negative selection, the monoclonal antibody mixture typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. In certain aspects, it may be desirable to enrich or positively select regulatory T cells that typically express CD4 ⁺ , CD25+, CD62Lhi, GITR+ and FoxP3 ⁺ . Alternatively, in certain aspects, regulatory T cells are depleted via anti-CD25 antibody-conjugated beads or other similar selection method.

In one embodiment, a T cell population may be selected that expresses one or more of IFN-γ, TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin, or other relevant molecules, such as other cytokines. Methods for screening for cell expression can be determined, for example, using the methods described in the publication according to PCT No: WO 2013/126712.

Cell concentration and surface (eg, particles such as beads) can be varied to select the desired cell population through positive or negative selection. In certain aspects, it may be desirable to substantially reduce the volume in which beads and cells are mixed together (eg, increasing cell concentration) to ensure maximum contact between cells and beads. For example, in one aspect, a concentration of 2 billion cells/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In a further aspect, a concentration of greater than 100 million cells/ml is used. In a further aspect, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml are used. In yet another aspect, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells/ml is used. IN

- 35 043737 additional aspects can use a concentration of 125 or 150 million cells/ml. The use of high concentrations may result in increased cell yield, cell activation, and cell expansion. Moreover, the use of high cell concentrations allows for more efficient capture of cells that may poorly express target antigens of interest, such as CD28-negative T cells, or from samples containing many tumor cells (eg, leukemia patient blood, tumor tissue etc.). Such cell populations may have therapeutic value and their production is desirable. For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which typically weakly express CD28.

In a related aspect, it may be desirable to use lower concentrations of cells. Significant dilution of the mixture of T cells and surface (eg, particles such as beads) minimizes interactions between particles and cells. This allows the selection of cells that express high amounts of the desired antigens that should bind to the particles. For example, CD4+ T cells express high levels of CD28 and can be captured more efficiently than CD8+ T cells at dilute concentrations. In one aspect, the cell concentration used is 5 x 10 ⁶ /ml. In other aspects, the concentration used may be from about 1x10 ⁵ /ml to 1x10 ⁶ /ml and any integer value in this range. In other aspects, cells can be incubated on a rotary shaker for periods of varying lengths at varying speeds at 2-10° C. or at room temperature.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and thawing step provides a more uniform product by removing granulocytes and to some extent monocytes from the cell population. After a washing step in which plasma and platelets are removed, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose , 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, then the cells are frozen to -80°C at a rate of 1 per minute and stored in the gas phase in a reservoir for storing liquid nitrogen. Other methods of controlled freezing can be used, as well as uncontrolled freezing directly at -20°C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to settle for one hour at room temperature before activation by the methods of the present invention.

It is also within the scope of the present invention to collect samples of blood or apheresis product from a subject during a period of time before expanded cells may be required, as described herein. In this regard, the source of cells to be expanded can be collected at any time needed, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions for which T-cell therapy will be effective for diseases such as those described herein. In one aspect, a blood sample or apheresis product is typically obtained from a healthy subject. In certain aspects, a blood sample or apheresis product is typically obtained from a healthy subject who is at risk of developing a disease, but who has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain aspects, T cells can be expanded, frozen, and used later. In certain aspects, samples are collected from a patient immediately after diagnosis of a particular disease, as described herein, but before the initiation of any treatment. In a further aspect, the cells are isolated from a blood sample or apheresis product from the subject prior to the initiation of any number of suitable treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressants such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation.

In a further aspect of the present invention, T cells are obtained from a patient immediately after treatment which leaves the subject with functional T cells. In this regard, it has been observed that after certain types of cancer treatments, in particular treatments using drugs that damage the immune system, soon after treatment during the period when patients usually recover from treatment, the quality of T

- 36 043737 cells may be optimal or improved with respect to their ability to reproduce ex vivo. Likewise, when manipulated ex vivo using the methods described herein, these cells may be in a favorable state for enhanced engraftment and expansion in vivo. Thus, it is within the scope of the present invention to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase. Moreover, in certain aspects, mobilization (eg, mobilization with GM-CSF) and symptomatic treatment regimens can be used to create a state in a subject in which the repopulation, recycling, regeneration and/or proliferation of specific cell types is beneficial, especially over a period of time. windows after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system. Activation and Expansion of T Cells T cells can be activated and expanded, typically using methods as described, for example, in US Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and 7,572,631.

In general, T cells of the present invention can be expanded by contacting a surface containing a substance attached thereto that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contacting an anti-CD3 antibody, or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or contacting a protein kinase activator With (for example, bryostatin) in combination with a calcium ionophore. To co-stimulate an accessory molecule on the surface of T cells, a ligand is used that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies may be used including 9.3, BT3, XR-CD28 (Diaclone, Besangon, France), as well as other methods well known in the art (Berg et al., Transplant Proc. 30(8):3975 -3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

T cells that have been stimulated for different periods of time may have different characteristics. For example, typical blood mononuclear cell products or peripheral blood mononuclear cell products after apheresis contain a population of helper T cells (TH, CD4 ⁺ ) that is larger than a population of cytotoxic or suppressor T cells (TC, CD8+). Expansion of T cells ex vivo through stimulation of CD3 and CD28 receptors results in a T cell population that, until approximately 8-9 days, consists predominantly of TH cells, whereas after approximately 8-9 days the T cell population contains an increasing population of TC cells . Accordingly, depending on the purpose of treatment, it may be preferable to infuse the subject with a population of T cells comprising predominantly TH cells. Likewise, if an antigen-specific subpopulation of TC cells has been isolated, it may be useful to expand this subpopulation to a larger number.

Moreover, in addition to CD4 and CD8 markers, other phenotypic markers change significantly during the cell proliferation process, but in a largely reproducible manner. Thus, this reproducibility provides the ability to tailor the activated T cell product to specific targets.

Once TFP is generated against mesothelin, various assays can be used to evaluate the activity of the molecule, such as, but not limited to, the ability to expand T cells following antigen stimulation, persistence of T cell expansion in the absence of restimulation, and anticancer activity in appropriate in vitro and animal models models. Assays to evaluate the effects of TFP against mesothelin are described in more detail below.

Western blot analysis of TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, for example, Milone et al., Molecular Therapy 17(8): 14531464 (2009)). Very briefly, T cells (a 1:1 mixture of CD4+ and CD8+ T cells) expressing TFP are expanded in vitro for over 10 days followed by lysis and SDS-PAGE under reducing conditions. TFP is detected by Western blotting using an antibody to the TCR chain. Similar subsets of T cells are used for SDS-PAGE analysis under non-reducing conditions to provide an assessment of covalent dimer formation.

In vitro expansion of TFP+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells is stimulated with alphaCD3/alphaCD28 and APC followed by transduction with lentiviral vectors expressing ZPB under the control of the promoters to be analyzed. Examples of promoters include the CMV IE, EF-1 alpha, ubiquitin C, or phosphoglycerokinase (PGA) gene promoters. Fluorescence of ZFB is estimated

- 37 043737 nivat on day 6 of culture in subsets of CD4+ and/or CD8+ T cells using flow cytometry (see, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Alternatively, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with alphaCD3/alphaCD28-coated magnetic beads on day 0 and transduced with TFP on day 1 using a bicistronic lintiviral vector expressing TFP together with USPB using a grooming sequence with ribosome 2A. Cultures were restimulated with mesothelin+ K562 cells (K562-mesothelin), wild-type K562 cells (K562 wild-type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562-BBL-3/28) after washing. Exogenous IL-2 is added to the cultures every other day at a concentration of 100 IU/ml. DFB+ T cells are counted by flow cytometry using bead-based counting (see, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). The sustained expansion of TFP+ T cells in the absence of restimulation can also be measured (see, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) was measured on day 8 of culture using a Coulter Multisizer III particle counter, following stimulation with alphaCD3/alphaCD28-coated magnetic beads on day 0 and transduction with the indicated TFP on day 1.

Animal models can also be used to measure TFP-T activity. For example, a xenograft model using human mesothelin-specific TFP+ T cells to treat cancer in immunodeficient mice (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). To describe it very briefly, once cancer is established, mice are randomized into treatment groups. Various numbers of engineered T cells are co-injected in a 1:1 ratio into cancer-bearing NOD/SCID/γ-/- mice. The copy number of each vector in spleen DNA obtained from mice was assessed at various time points after T cell injection. Animals are assessed for cancer at weekly intervals. Mesothelin+ peripheral blood cancer cell counts are measured in mice injected with alphamesothelin-zeta TFP+ T cells or mock-transduced T cells. Survival curves between groups were compared using the log-rank test. In addition, the absolute values of peripheral blood CD4+ and CD8+ T cells 4 weeks after T cell injection into NOD/SCID/γ-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP using a bicistronic lintiviral vector that encodes TFP bound to USPB. T cells are normalized to 45-50% of the injected GFB+ T cells by mixing with mock-transduced cells before injection and confirmed by flow cytometry. Animals are assessed for cancer at weekly intervals. Survival curves among TFP+ T cell groups were compared using the log-rank test.

The dose-dependent response to TFP treatment can be assessed (see, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). For example, peripheral blood is obtained 35-70 days after cancer establishment from mice injected on day 21 with TFP-T cells, an equivalent number of mock-transduced T cells, or non-injected T cells. Mice from each group were randomly bled to determine the number of mesothelin+ peripheral blood cancer cells, and then the animals were sacrificed on days 35 and 49. The remaining animals are assessed on days 57 and 70.

Assessment of cell proliferation and cytokine production has been described previously, for example, in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of TFP-mediated proliferation is performed on microtiter plates by mixing washed T cells with cells expressing mesothelin or CD32 and CD137 (KT32-BBL) to obtain a final ratio of T cells to mesothelin expressing cells of 2:1 . Cells expressing mesothelin are irradiated with gamma radiation before use. Monoclonal antibodies to CD3 (clone OKT3) and CD28 (clone 9.3) are added to cultures with KT32-BBL cells as a positive control to stimulate T cell proliferation, as these signals support long-term expansion of CD8+ T cells ex vivo. T cells in cultures were enumerated using CountBright™ fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer. TFP+ T cells are identified by expression of USFB using T cells that have been constructed using TFP-expressing lentiviral vectors linked to USFB-2A. For TFP+ T cells that do not express ZPB, TFP+ T cells are detected using biotinylated recombinant mesothelin protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells is also simultaneously determined using specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours after restimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according to the manufacturer's instructions. Fluorescence was assessed using a FACScalibur flow pitometer and data analyzed according to the manufacturer's instructions. Cytotoxicity can be assessed using a standard ⁵¹ Cr release assay (see, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). In short, cells

- 38 043737 targets are loaded with ⁵¹ Cr (as NaCrO ₄ , New England Nuclear) at 37°C for 2 hours with frequent stirring, washed twice in complete RPMI medium and plated on microtiter plates. Effector T cells are mixed with target cells in wells in complete RPMI medium at various ratios of effector cells to target cells (E:T). Additional wells are also prepared containing only the medium (spontaneous release, SR) or a 1% solution of Triton-X 100 detergent (complete release, PT). After 4 hours of incubation at 37°C, the supernatant was collected from each well. The released ⁵¹ Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is performed in at least three replicates, and the percentage of lysis is calculated using the following formula: % lysis = (EV-DM)/(PV-DM), where EV represents the average value of ⁵¹ Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific TFP migration and proliferation in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/yc−/− (NSG) mice were injected i.v. with cancer cells, followed 7 days later by T cells 4 h after electroporation with TFP constructs. T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single injection of TFP+ T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail vein injection of T cells electroporated with TFP mesothelin via 7 days. Animals are imaged at various time points after injection. For example, photon density heat maps of firefly luciferase-positive cancers can be obtained from representative mice on day 5 (2 days before treatment) and day 8 (24 hours after TFP+ peripheral blood leukocytes). Other assays may also be used to evaluate the anti-mesothelin TFP constructs of the present invention, including those described in the Examples section of this document, as well as assays known in the art.

Therapeutic use.

Diseases and/or disorders associated with mesothelin.

In one aspect, the present invention provides methods for treating a disease associated with mesothelin expression. In one aspect, the present invention provides methods for treating a disease wherein a portion of the tumor is mesothelin negative and a portion of the tumor is mesothelin positive. For example, the TFP of the present invention is useful for treating subjects who have received treatment for a disease associated with increased mesothelin expression, wherein the subject who has received treatment for increased levels of mesothelin exhibits a disease associated with increased levels of mesothelin. In one aspect, the present invention provides a vector comprising anti-mesothelin TFP operably linked to a promoter for expression in mammalian T cells. In one aspect, the present invention provides a recombinant T cell expressing mesothelin TFP for use in the treatment of mesothelin-expressing tumors, wherein the recombinant T cell expressing mesothelin TFP is referred to as mesothelin TFP-T. In one aspect, the mesothelin TFP-T of the present invention is capable of contacting a tumor cell with at least one mesothelin TFP of the present invention expressed on its surface such that the TFP-T targets the tumor cell and tumor growth is inhibited.

In one aspect, the present invention provides a method of inhibiting the growth of a mesothelin-expressing tumor cell, comprising contacting the tumor cell with a mesothelin TFPT cell of the present invention such that TFP-T is activated in response to an antigen and targets the cancer cell, causing the tumor to grow inhibited.

In one aspect, the present invention relates to a method of treating cancer in a subject. The method includes administering to a subject a TFP-T cell mesothelin of the present invention such that a cancer is treated in the subject. An example of a cancer to be treated with the mesothelin TFP-T cell of the present invention is a cancer associated with mesothelin expression. In one aspect, the cancer is mesothelioma. In one aspect, the cancer is pancreatic cancer. In one aspect, the cancer is ovarian cancer. In one aspect, the cancer is gastric cancer. In one aspect, the cancer is lung cancer. In one aspect, the cancer is endometrial cancer. In some embodiments, TFP mesothelin therapy may be used in combination with one or more additional treatments. The present invention includes a type of cell therapy in which T cells are genetically modified to express TFP, and the TFP-expressing T cell is administered to a recipient in need thereof. The injected cell is capable of destroying tumor cells in the recipient. Unlike antibody therapy, TFP-expressing T cells are capable of replication in vivo, resulting in

- 39 043737 long-lasting, which can lead to sustainable tumor control. In various aspects, the T cells or progeny thereof administered to the patient are retained in the patient for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years or five years after the T cell was administered to the patient. The present invention also includes a type of cell therapy in which T cells are modified, for example, with in vitro transcribed RNA, to transiently express TFP, and the TFP-expressing T cell is administered to a recipient in need thereof. The injected cell is capable of destroying tumor cells in the recipient. Thus, in various aspects, T cells administered to a patient are retained for less than one month, such as three weeks, two weeks, or one week, after the T cell is administered to a patient. Without being bound by any particular theory, the antitumor immune response elicited by TFP-expressing T cells may be an active or passive immune response or, alternatively, may be due to a direct or indirect immune response. In one aspect, TFP-transduced T cells exhibit specific secretion of proinflammatory cytokines and potent cytolytic activity in response to human cancer cells expressing mesothelin antigen, resist inhibition by soluble mesothelin, mediate nonspecific cytolysis, and/or mediate regression of established human tumors. For example, antigen-null tumor cells within a heterogeneous field of a mesothelin-expressing tumor may be susceptible to indirect destruction by mesothelin-redirected T cells that previously antagonized adjacent antigen-positive cancer cells. In one aspect, the human TFP-modified T Cells of the present invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is human.

For ex vivo immunization, at least one of the following occurs in vitro before the cell is administered to the mammal: i) cell proliferation; ii) introducing a nucleic acid encoding TFP into the cells, or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are described in more detail below. Briefly, cells are isolated from a mammal (eg, human) and genetically modified (ie, transduced or transfected in vitro) with a TFP expression vector described herein. The TFP-modified cell can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be human, and the TFP-modified cell may be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic to the recipient.

A procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in US Pat. No. 5,199,942, incorporated herein by reference, and may be applied to the cells of the present invention. Other suitable methods are known in the art, so the present invention is not limited to any particular method of ex vivo cell expansion. Briefly, ex vivo T cell culture and expansion involves: (1) isolating CD34+ hematopoietic stem cells and progenitor cells from a mammal from peripheral blood or bone marrow explants; and (2) expansion of such cells ex vivo. In addition to the cellular growth factors described in US Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand can be used for cell culture and propagation.

In addition to the use of a cell-based vaccine for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

In general, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the TFP-modified T cells of the present invention are used to treat diseases, disorders and conditions associated with mesothelin expression. In some aspects, the T cells of the present invention are used to treat patients at risk of developing diseases, disorders and conditions associated with mesothelin expression. Thus, the present invention provides methods for treating or preventing diseases, disorders and conditions associated with mesothelin expression, comprising administering to a subject in need thereof a therapeutically effective amount of the TFP-modified T cells of the present invention.

In one aspect, the TFP T cells of the present invention can be used to treat a proliferative disease, such as cancer, or a malignant tumor, or a precancerous condition. In one aspect, the cancer is mesothelioma. In one aspect, the cancer is pancreatic cancer. In one aspect, the cancer is ovarian cancer. In one aspect, cancer is

- 40 043737 is stomach cancer. In one aspect, the cancer is lung cancer. In one aspect, the cancer is endometrial cancer. Moreover, disease associated with the expression of mesothelin includes, but is not limited to, for example, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing mesothelin. Non-cancer related indications associated with mesothelin expression include, but are not limited to, autoimmune disease (eg, lupus), inflammatory diseases (allergy and asthma), and transplantation.

The TFP-modified T cells of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. The present invention also provides methods for inhibiting the proliferation or reduction of a population of mesothelin-expressing cells, the methods comprising contacting a population of cells containing mesothelin-expressing cells with an anti-mesothelin TFP-T cell of the present invention that binds to the mesothelin-expressing cell . In a specific aspect, the present invention provides methods for inhibiting proliferation or reducing a population of cancer cells expressing mesothelin, the methods comprising contacting a population of cancer cells expressing mesothelin with an anti-mesothelin TFP-T cell of the present invention that binds to mesothelin- expressing cell. In one aspect, the present invention provides methods for inhibiting proliferation or reducing a population of cancer cells expressing mesothelin, the methods comprising contacting a population of cancer cells expressing mesothelin with an anti-mesothelin TFP-T cell of the present invention that binds to mesothelin- expressing cell. In some aspects, the anti-mesothelin TFP-T cell of the present invention reduces the number, number, number or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject or animal model with a cancer associated with mesothelin-expressing cells, according to relative to negative control. In one aspect the subject is a person.

The present invention also provides methods for preventing, treating and/or controlling a disease associated with mesothelin-expressing cells (e.g., mesothelin-expressing cancer), the methods comprising administering to a subject in need an anti-mesothelin TFP-T cell of the present invention, which binds to mesothelin-expressing cells. In one aspect the subject is a person. Non-limiting examples of disorders associated with mesothelin-expressing cells include autoimmune diseases (such as lupus), inflammatory diseases (such as allergies and asthma), and cancers (such as pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, or endometrial, or atypical cancers expressing mesothelin).

The present invention also provides methods for preventing, treating and/or controlling a disease associated with mesothelin-expressing cells, the methods comprising administering to a subject in need an anti-mesothelin TFP-T cell of the present invention that binds to the mesothelin-expressing cell. In one aspect the subject is a person.

The present invention provides methods for preventing the recurrence of cancer associated with mesothelin-expressing cells, the methods comprising administering to a subject in need thereof an anti-mesothelin TFP-T cell of the present invention that binds to the mesothelin-expressing cell. In one aspect, the methods include administering to a subject in need thereof an effective amount of an anti-mesothelin TFP-T cell described herein that binds to a mesothelin-bmca-expressing cell, in combination with an effective amount of another treatment. The TFP-expressing cell combination therapy described herein may be used in combination with other known agents and therapies. Administration in combination, as used herein, means that two (or more) different treatments are delivered to a subject during the course of a disease, e.g., two or more treatments are delivered after the subject has been diagnosed with a disorder and before the disorder was cured, or eliminated, or treatment was stopped for other reasons. In some embodiments, delivery of one treatment is still occurring when delivery of the second treatment begins, thus there is overlap in terms of administration. This is sometimes referred to herein as simultaneous or collateral delivery. In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments, treatment is more effective in either case due to combination administration. For example, the second treatment is more effective, for example, an equivalent effect is observed with a lower dose of the second treatment, or the second treatment reduces symptoms to a greater extent than observation

- 41 043737 would be the case if the second treatment was administered in the absence of the first treatment, or a similar situation was observed for the first treatment. In some embodiments, delivery is such that the reduction in a symptom or other disease-related parameter is greater than would be observed if one treatment were delivered in the absence of the other. The effect of two treatments may be partially additive, fully additive, or greater than additive. Delivery may be carried out in such a way that the effect of the first treatment agent administered will still be determined at the time of delivery of the second agent.

In some embodiments, the at least one additional therapeutic agent includes a TFP-expressing cell. Also provided are T cells that express multiple TFPs that bind to the same or different target antigens or to the same or different epitopes on the same target antigen. Also provided are T cell populations in which a first T cell subpopulation expresses a first TFP and a second T cell subpopulation expresses a second TFP.

The TFP-expressing cell described herein and at least one additional therapeutic agent may be administered simultaneously in the same or separate compositions, or administered sequentially. In the case of sequential administration, the TFP-expressing cell described herein may be administered first and the additional substance may be administered second, or the order of administration may be reversed.

In additional aspects, a TFP-expressing cell described herein can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immunosuppressive agents such as such as alemtuzumab, anti-CD3 or other antibody therapies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. The TFP-expressing cell described herein can also be used in combination with a peptide vaccine such as that described by Izumoto et al. 2008 J Neurosurg 108:963-971. In an additional aspect, the TFP-expressing cell described herein can also be used in combination with a myeloid cell differentiation promoter (e.g., all-trans retinoic acid), an inhibitor of myeloid-derived suppressor cell (MDSC) proliferation (e.g., c-kit receptor inhibitors or a VEGF inhibitor), inhibition of MDSC function (eg, COX2 inhibitors or phosphodiesterase-5 inhibitors), or therapeutic elimination of MDSCs (eg, through chemotherapy regimens such as doxorubicin and cyclophosphamide treatment). Other therapeutic agents that can prevent MDSC proliferation include aminobisphosphonate, bisphosphonate, sildenafil and tadalafil, nitroaspirin, vitamin D3, and gemcitabine. (See, for example, Gabrilovich and Nagaraj, Nat. Rev. Immunol, (2009) v9(3): 162-174).

In one embodiment, the subject may be administered a substance that reduces or alleviates the side effect associated with administration of the TFP-expressing cell. Side effects associated with administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS) and hemophagocytic lymphohistiocytosis (HLH), also called macrophage activation syndrome (MAS). Symptoms of CRS include fever, nausea, transient hypotension, hypoxia, etc. Accordingly, the methods described herein may include administering a TFP-expressing cell described herein to the subject and additionally administering an agent to control increased levels of soluble factor resulting from treatment with a TFP-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-γ, TNFa, IL-2, IL-6, and IL8. Thus, the substance administered to treat this side effect may be a substance that neutralizes one or more of these soluble factors. Such substances include, but are not limited to, a steroid, a TNFa inhibitor, and an IL-6 inhibitor. An example of a TNFa inhibitor is entanercept. An example of an IL-6 inhibitor is tocilizumab (toc). In one embodiment, the subject may be administered a substance that increases the activity of a TFP-expressing cell. For example, in one embodiment, the substance may be one that inhibits an inhibitory molecule. Inhibitory molecules, such as programmed cell death protein 1 (PD1), may in some embodiments reduce the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition of an inhibitory molecule, for example by inhibiting DNA, RNA or protein level, can optimize the efficiency of the TFP-expressing cell. In embodiments, an inhibitory nucleic acid, such as an inhibitory nucleic acid, such as dsRNA, such as siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in a TFP-expressing cell. In one embodiment, the inhibitor is an shRNA. IN

- 42 043737 In one embodiment, the inhibitory molecule is inhibited within a TFP-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is coupled to a nucleic acid that encodes a component, eg, all components, of TFP. In one embodiment, an inhibitor of an inhibitory signal may be, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the substance may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (for example, ipilimumab (also called MDX-010 and MDX-101 and marketed as Yervoy™; Bristol-Myers Squibb; tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206)). In one embodiment, the substance is an antibody or antibody fragment that binds to TIM3. In one embodiment, the substance is an antibody or an antibody fragment that binds to LAG3.

In some embodiments, T cells can be modified (eg, by gene transfer) in vivo using a lentivirus, such as a lentivirus that specifically targets a CD4+ or CD8+ T cell. (See, for example, Zhou et al., J. Immunol. (2015) 195:2493-2501).

In some embodiments, the substance that enhances the activity of a TFP-expressing cell may be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule or fragment thereof and the second domain is a polypeptide that is associated with a positive signal, for example, a polypeptide containing an intracellular signaling domain, as described herein. In some embodiments, the polypeptide that is associated with a positive signal may include a CD28, CD27, ICOS co-stimulatory domain, e.g., a CD28, CD27, and/or ICOS intracellular signaling domain, and/or a primary signaling domain, e.g., CD3 zeta, e.g. as described herein. In one embodiment, the fusion protein is expressed by the same cell that expresses TFP. In another embodiment, the fusion protein is expressed by a cell, for example a T cell, that does not express anti-mesothelin TFP.

In some embodiments, a domain of a human or humanized antibody comprising an antigen binding domain that is a nucleic acid-encoded anti-mesothelin binding domain, or an antibody comprising an anti-mesothelin binding domain, or a cell expressing a nucleic acid-encoded anti-mesothelin binding domain is affinities as a maximum of about 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 pM; and/or at least about 100 nM, 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM; and/or about 200 nM, 100 nM, 75 nM, and 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM , 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM.

Pharmaceutical compositions.

The pharmaceutical compositions of the present invention may contain a TFP-expressing cell, for example, a plurality of TFP-expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide) and preservatives. The compositions of the present invention, in one aspect, are formulated for intravenous administration.

The pharmaceutical compositions of the present invention can be administered in a manner that is appropriate to the disease being treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate doses may be established in clinical studies.

In one embodiment, the pharmaceutical composition contains substantially no, e.g., no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual CD3/CD28 antibody-coated beads, mouse antibodies, mixed human serum, bovine serum albumin, bovine serum, culture medium components, cell vector packaging or plasmid components, and bacteria and fungi. In one embodiment, the bacterium is at least one

- 43 043737 brane from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilias influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia and Streptococcus pyogenes group A. When an immunologically effective amount is indicated, an antitumor effective amount, inhibit common tumor the effective amount or therapeutic amount, the exact amount of the composition of the present invention to be administered, can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). In general, it may be noted that a pharmaceutical composition containing the T cells described herein can be administered in doses of 10 ⁴ to 10 ⁹ cells/kg body weight, in some cases 10 5 to 10 ⁶ cells/kg body weight , including all integer values in these ranges. The T cell compositions may also be administered multiple times at these dosages. Cells can be administered using infusion techniques that are well known in the field of immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

In some aspects, it may be necessary to administer activated T cells to a subject, then re-draw blood thereafter (or perform apheresis), activate the resulting T cells in accordance with the present invention, and re-infusion the activated and expanded T cells into the patient. This process may be repeated several times every few weeks. In some aspects, T cells can be activated in a blood sample as small as 10 cc. cm up to 400 cc cm. In some aspects, T cells are activated in a 20 cc blood sample. cm, 30 cu. cm, 40 cu. cm, 50 cu. cm, 60 cu. cm, 70 cu. cm, 80 cu. cm, 90 cu. cm or 100 cc. cm.

Administration of the compositions to the subject may be accomplished by any convenient means, including aerosol inhalation, injection, oral administration, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by intravenous injection. The T cell compositions may be injected directly into a tumor, lymph node, or site of infection.

In a particular illustrative aspect, a subject may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate cells of interest, such as T cells. Such T cell isolates can be expanded by methods known in the art and processed such that one or more TFP constructs of the present invention can be introduced, thereby creating a TFP-expressing T cell of the present invention. Subjects in need may subsequently undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some aspects, following or concurrently with transplantation, subjects are infused with expanded TFP T cells of the present invention. In a further aspect, the expanded cells are administered before or after surgery.

The dosage of the above treatments requiring administration to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed in accordance with accepted practices in the art. The dose of alemtuzumab, for example, will typically range from 1 to about 100 mg for adult patients, typically administered daily for a period of 1 to 30 days. The preferred daily dose is 1 to 10 mg per day, although higher doses of up to 40 mg per day may be used in some cases (described in US Pat. No. 6,120,766).

In one embodiment, TFP is introduced into T cells, for example, using in vitro transcription, and the subject (e.g., a human) receives an initial administration of TFP-T cells according to the present invention, as well as one or more subsequent administrations of TFP-T cells according to the present invention, wherein one or more subsequent administrations are performed less than 15 days, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, a subject (eg, a human) receives more than one administration of the TFP-T cells of the present invention per week, such as 2, 3, or 4 administrations of the TFP-T cells of the present invention per week. In one embodiment, a subject (eg, a human) is given more than one TFP-T cell administration per week (eg, 2, 3, or 4 administrations per week) (also referred to herein as a cycle) followed by a break in TFP-T-cell administration. cells for one week, and then the subject receives one or more additional TFP-T cell administrations (eg, more than one TFP-T cell administration per week). In another embodiment, a subject (eg, a human) is subjected to more than one cycle of TFP-T cells and the time period between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, TFP-T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP-T cells of the present invention are administered for at least two, three, four, five, six, seven, eight or more weeks. In one aspect, anti-mesothelin TFP-T cells are produced using lentiviral viral vectors such as

- 44 043737 lentivirus. The TFP-T cells thus obtained will have robust TFP expression.

In one aspect, TFP-T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10.

11, 12, 13, 14, 15 days after transduction. Transient expression of TFP can be accomplished by delivery of a TFP RNA vector. In one aspect, TFP RNA is transduced into a T cell by electroporation.

A potential problem that may arise in patients treated with T cells transiently expressing TFP (particularly with murine TFP-T cells bearing scFv) is anaphylaxis after multiple treatments.

Without being limited by theory, it is believed that such an anaphylactic response may be caused by the patient developing an anti-TFP humoral response, i.e., anti-TFP antibodies of the anti-IgE isotype. It is believed that the patient's antibody-producing cells undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype when there is a gap of ten to fourteen days in exposure to the antigen.

If a patient is at high risk of developing an anti-TFP antibody response during treatment with transient TFP therapy (eg, derived by RNA transduction), interruptions in TFP-T cell infusion should not last more than ten to fourteen days.

Examples

The invention will now be described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and should not be interpreted as limiting unless otherwise noted. Thus, the invention should not be taken to be limited to the following examples, but rather to be taken to embrace any and all variations that will become apparent as a result of the ideas presented herein. Without further description, it is believed that using the foregoing description and the following illustrative examples, one skilled in the art can prepare and use the compounds of the present invention and practice the claimed methods. The following demonstrative examples specifically indicate various aspects of the present invention and should not be interpreted as limiting in any way the remainder of the description.

Example 1: TFP designs.

Anti-mesothelin TFP constructs are created by cloning an anti-mesothelin scFv DNA fragment linked to a CD3 or TCR DNA fragment via a DNA sequence encoding a short linker (CL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:2) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE ( SEQ ID NO:3), into the p510 vector ((System Biosciences (SBI)) at the XbaI and EcoRI sites.

The resulting anti-mesothelin TFP constructs are as follows: p510_antimesothelin_LL_TCRa (scFv anti-mesothelin - long linker - human full-length T-cell receptor a-chain), p510_antimesothelin_LL_TCR aC (scFv anti-mesothelin - long linker human T-cell receptor constant domain a-chain ), p510_antimesothelin_LL_TCRe ( scFv against mesothelin - long linker - human full-length β-chain of the T-cell receptor), p510_antimesothelin_LL_TCReC (scFv against mesothelin - long linker - human β-chain of the constant domain of the T-cell receptor), p510_antimesothelin_LL_CD3y (scFv against mesothelin long linker - human CD3y chain ), p510_antimesothelin_LL_CD35 (scFv against mesothelin long linker - human CD35 chain), p510_antimesothelin_LL_CD3ε (scFv against mesothelin long linker - human CD3ε chain), p510_antimesothelin_SL_TCRe (scFv against mesothelin short linker - human full size T cell receptor β chain), p510_antimesothe lin_SL_CD3y (scFv against mesothelin - short linker - human chain CD3y), p510_antimesothelin_SL_CD35 (scFv against mesothelin - short linker - human chain CD35), p510_antimesothelin_SL_CD3ε (scFv against mesothelin - short linker - human chain CD3ε).

The anti-mesothelin CAR construct p510_antimesothelin_28Z is obtained by cloning the synthesized DNA encoding anti-mesothelin, the partial extracellular domain of CD28, the transmembrane domain of CD28, the intracellular domain of CD28 and CD3 zeta into the p510 vector at the XbaI and EcoRI sites.

Example 2: Antibody sequences. Obtaining antibody sequences.

The canonical polypeptide sequence of human mesothelin can be found under UniProt accession number Q13421 (or Q13421-1). Antibody polypeptides are provided that are capable of specifically binding to human mesothelin polypeptide, as well as fragments or domains thereof. Antibodies to mesothelin can be obtained using various methods (see, for example, Nicholson et al, 1997). When mouse anti-mesothelin antibodies are used as starting material, humanization of mouse anti-mesothelin antibodies is desirable in clinical settings where mouse-specific residues can induce a human anti-mouse antigen (HAMA) response in subjects receiving T-cell receptor fusion protein (TFP) treatment (TCR), i.e. treatment with T cells transduced with TFP.mesothelin constructs. Humanization

- 45 043737 is carried out by grafting murine anti-mesothelin antibody CDR regions onto corresponding human germline acceptor scaffolds, optionally including other modifications of the CDR regions and/or framework regions. As presented herein, numbering of antibody or antibody fragment residues is done according to Kabat (Kabat E. A. et al, 1991; Chothia et al,

1987).

Preparation of scFv.

Human or humanized anti-mesothelin IgGs are used to obtain scFv sequences for TFP constructs. DNA sequences encoding human or humanized V _L and V _H domains are obtained, and the codons for the constructs are optionally optimized for expression in Homo sapiens cells. The order in which the V _L and V H domains appear in scFv is different (i.e., V _L -V _H or V _H -V _L orientation), and three copies of the G ₄ S or G ₄ S subunit (G ₄ S) ₃ join the variable domains to create the scFv domain. Anti-mesothelin scFv plasmid constructs may have optional Flag, His, or other affinity tags, and the constructs are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include FACS binding analysis, Proteon kinetic analysis, and staining of mesothelin-expressing cells.

Examples of V _L and V _H domains against mesothelin, CDRs and nucleotide sequences encoding them can be described in US patent Nos.: 9,272,002; 8,206,710; 9,023,351; 7,081,518; 8,911,732; 9,115,197 and 9,416,190; and US Patent Publication No. 20090047211. Other examples of the anti-mesothelin V _L and V _H domains, CDRs, and nucleotide sequences encoding them, respectively, may include the domains, CDRs, and sequences of the following monoclonal antibodies: rat anti-mesothelin antibody 420411, rat anti-mesothelin antibody 420404, mouse anti-mesothelin antibody MN-1, mouse anti-mesothelin antibody MB-G10, mouse anti-mesothelin antibody ABIN233753, rabbit anti-mesothelin antibody FQS3796(3), rabbit anti-mesothelin antibody TQ85, mouse anti-mesothelin antibody TA307799, rat antibody to mesothelin 295D , rat anti-mesothelin antibody B35, mouse anti-mesothelin antibody 5G157, mouse anti-mesothelin antibody 129588, rabbit anti-mesothelin antibody 11C187, mouse anti-mesothelin antibody 5B2, rabbit anti-mesothelin antibody SP74, rabbit anti-mesothelin antibody D4X7M, mice new antibody to mesothelin C-2 , mouse anti-mesothelin antibody C-3, mouse anti-mesothelin antibody G-1, mouse anti-mesothelin antibody G-4, mouse anti-mesothelin antibody K1, mouse anti-mesothelin antibody B-3, mouse anti-mesothelin antibody 200-301-A87, mouse anti-mesothelin antibody 200-301-A88, rabbit anti-mesothelin antibody EPR2685(2), rabbit anti-mesothelin antibody EPR4509, or rabbit anti-mesothelin antibody PPI-2e(IHC).

In some embodiments, single domain (V _HH ) binding antibodies are used, such as those set forth in SEQ ID NOs 58, 59 and 55 (SD1, SD4 and SD6, respectively). Sources of TCR subunits

All subunits of the human T cell receptor (TCR) complex contain an extracellular domain, a transmembrane domain, and an intracellular domain. The human TCR complex contains CD3 epsilon polypeptide, CD3 gamma polypeptide, CD3 delta polypeptide, CD3 zeta polypeptide, TCR alpha chain polypeptide and TCR beta chain polypeptide. The canonical human CD3 epsilon polypeptide sequence can be found under Uniprot accession number P07766. The canonical human CD3-gamma polypeptide sequence can be found under Uniprot accession number P09693. The canonical human CD3-delta polypeptide sequence can be found under Uniprot accession number P043234. The canonical human CD3-zeta polypeptide sequence can be found under Uniprot accession number P20963. The canonical human TCR alpha chain sequence can be found under Uniprot accession number Q6ISU1. The canonical sequence of the C region of the human TCR beta chain can be found under Uniprot accession number P01850, the sequence of the V region of the human TCR beta chain - under the number P04435.

Canonical human CD3 epsilon polypeptide sequence: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDK NIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDV MSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQ RGQNKERPPPVPNPDYEPIR KGQRDLYSGLNQRRI (SEQ ID NO:4).

Canonical human CD3-gamma polypeptide sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGF LTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFV LAVGVYFIAGQDGVRQSRASDKQTLLP NDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:5).

Canonical human CD3 delta polypeptide sequence: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDP RGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGR LSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS ( SEQ ID NO:6). Canonical human CD3-zeta polypeptide sequence:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQ

- 46 043737

GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:7).

Canonical human TCR alpha chain sequence:

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFS AGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGE ASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPATTTRLRALGSH RLHPATETGGREATSSPRPQPR DRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSALR APSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:8).

The canonical sequence of the human TCR alpha chain C region is:

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF NLLMTLRLWSS (SEQ ID NO:9).

Canonical sequence of the human TCR alpha chain V region CTL-L17:

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYK KYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAKGAGTASKLTF GTGTRLQVTL (SEQ ID NO: 10).

The canonical sequence of the human TCR beta chain C region is:

EDLNKVFPPEVAVAVAVFEPSEISHTQKATLVCLVCLSWVNGKEVSGVSTDPQPLKPALNDSRVSATFWHFRCQFYGLSEVTQVTQVSAWSREVSALYSEVSRAKVSEAWS RADAWG RADAWG RADAWG Radawg Radawg Rad FTSVSYQGVLSATILLGKATLAVLVSALVLMAMVKRKDF (SEQ ID NO: 11).

Canonical sequence of human TCR beta chain V region CTL-L17:

MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQHFGDGTRLSIL (SEQ ID NO:12).

Canonical sequence of the human TCR beta chain V region YT35:

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSANYGYTFGSGTRLTVV (SEQ ID NO: 13).

Derivation of TFP from TCR and scFv domains.

mesothelin scFvs are recombinantly linked to CD3 epsilon or other TCR subunits (see 1C) via a linker sequence such as G ₄ S, (G ₄ S) ₂ (G ₄ S) ₃ or (G ₄ S) ₄ . Various linkers and scFv configurations are used. TCR alpha and beta chains have been used to produce TFP as full-length polypeptides or only their constant domains. Any variable sequence of the TCR alpha and beta chain can be used to create TFP. TFP expression vectors

Expression vectors are provided that include: a promoter (the cytomegalovirus (CMV) enhancer promoter), a secretion signal sequence, a polyadenylation signal and a transcription terminator (the bovine growth hormone gene (BGH)), an episomal replication element and replication in prokaryotes (eg, with the origin of SV40 and ColE1 or others known in the art), and elements for providing selection (ampicillin resistance gene and zeocin marker). Preferably, the nucleic acid construct encoding TFP is cloned into a lentiviral expression vector, and expression is confirmed based on the quantity and quality of the TFP.mesothelin-transduced T cell effector response (mesothelin.TFP, or mesothelin.TFP T cells, or TFP.mesothelin , or TFP.mesothelin T cells) in response to mesothelin+ target cells. T cell effector responses include, but are not limited to, cell expansion, proliferation, duplication, cytokine production, and target cell lysis or cytolytic activity (i.e., degranulation).

TFP.mesothelin lentiviral transfer vectors are used to generate genomic material packaged into pseudotyped VSV-G lentiviral particles. The lentiviral transfer vector DNA is mixed with the three packaging components VSV-G, gag/pol and rev in combination with Lipofectamine® reagent to transfect them together into HEK-293 (fetal kidney, TCC® CRL-1573™) cells. After 24 and 48 hours, the medium is collected, filtered and concentrated by ultracentrifugation. The resulting viral preparation is stored at -80°C. The number of transduced units is determined by titration on Sup-T1 cells (T-cell lymphoblastic lymphoma, ATCC® CRL-1942™). Redirected TFP.mesothelin T cells are produced by activating fresh naïve T cells, for example with anti-CD3 and anti-CD28 beads for 24 hours, and then adding the appropriate number of transduced units to obtain the desired percentage of transduced T cells. These modified T cells are left to proliferate until they settle and shrink in size, at which point they are cryopreserved for later analysis. Cell numbers and sizes are measured using a Coulter Multisizer™ III. Before cryopreservation, the percentage of transduced cells (expressing TFP.mesothelin on the cell surface) and the relative fluorescence intensity of such expression are determined by flow cytometry analysis. The histogram examines the relative levels of TFP expression by comparing the percentage of transduced cells with their relative fluorescence intensity. In some embodiments, multiple TFPs are administered by T cell transduction using multiple viral vectors.

Evaluation of cytolytic activity, proliferation capabilities and cytokine secretion of humanized redirected TFP-T cells.

The functionality of TFP.mesothelin T cells to produce cell surface expressed TFP as well as target tumor cell killing, proliferation and cytokine secretion is determined using assays known in the art. Human peripheral blood mononuclear cells (PBMCs, e.g., apheresis blood from a normal donor whose naïve T cells were obtained by negative selection of T cells, CD4+ and CD8+ lymphocytes) are treated with human interleukin-2 (IL-2), then activated with beads against CD3 and CD28, for example, in 10% RPMI at 37°C, 5% CO ₂ before transduction with lentiviral vectors encoding TFP. Flow cytometry assays are used to confirm the presence of TFP on the cell surface, for example using an anti-FLAG antibody or an anti-mouse variable domain antibody. Cytokine production (eg, IFN-γ) is measured using ELISA or other assays.

Example 3: Efficacy of human TFP-T cells in a mouse model of human ALL.

Primary human ALL cells can be grown in immunocompromised mice (eg, NSG or NOD) without the need for in vitro culture.

Likewise, cultured human ALL cell lines can induce leukemia in such mice. Mice with ALL can be used to test the effectiveness of human TFP.mesothelin T cells, for example in the HALLX5447 model. The measured value of this model is the survival of mice following intravenous (IV) infusion of ALL cells in the absence and presence of IV human TFP.mesothelin T cells.

Example 4: Demonstration of multiplexed TFP polypeptides and use of multiplexed humanized redirected TFP-T cells.

The TFP polypeptides provided herein are capable of operably binding to endogenous TCR subunit polypeptides to form functional TCR complexes. Here, multiple TFPs are used in lentiviral vectors to transduce T cells to create a functional multiplex recombinant TCR complex. For example, a T cell is provided comprising: i) a first TFP having an extracellular domain, a transmembrane domain and an intracellular domain from a CD3 delta polypeptide and a mesothelin-specific scFv antibody fragment, and ii) a second TFP having an extracellular domain, a transmembrane domain and intracellular domain from CD3-gamma polypeptide and mesothelin-specific antibody fragment. The first TFP and the second TFP are capable of interacting with each other as well as with endogenous TCR subunit polypeptides, thereby forming a functional TCR complex.

The use of these TFP.mesothelin multiplex humanized T cells can be demonstrated in liquid tissue tumors and solid tumors, as presented in Examples 2 and 3 above.

Example 5: Preparation of T cells transduced with TFP.

Preparation of lentiviruses.

Lentiviruses encoding the corresponding constructs are prepared as follows.

5 x 10 ⁶ HEK-293F T cells are seeded per 100 mm dish and left overnight to achieve 70-90% confluency. 2.5 μg of the indicated DNA plasmids and 20 μl of Lentivirus Packaging Mix (ALSTEM, Cat. No. VP100) are diluted in 0.5 ml of DMEM or Opti-MEM® I medium without serum and mixed gently. In a separate tube, 30 µl of NanoFect® Transfection Reagent (ALSTEM, cat. no. NF100) is diluted in 0.5 ml of DMEM or Opti-MEM® I without serum and mixed gently. The NanoFect/DMEM and DNA/DMEM solutions are then mixed together and vortexed for 10-15 seconds before incubating the DMEM-NanoFect plasmid mixture at room temperature for 15 minutes. The entire transfection complex from the previous step is added dropwise to the cell plate and rocked to disperse the transfection complex evenly throughout the plate. The plate is then incubated overnight at 37°C in a humidified incubator with 5% CO2. The next day, the supernatant is replaced with 10 ml of fresh medium and 20 µl of ViralBoost (500x, ALSTEM, cat. no. VB100) is added. The plates are then incubated at 37°C for an additional 24 hours. The lentivirus containing the supernatant is then collected into a sterile 50 ml capped conical centrifuge tube and placed on ice. After centrifugation at 3000 rpm for 15 minutes at 4°C, the released supernatant is filtered using a sterile low protein binding filter with a pore diameter of 0.45 μm, and the virus is subsequently isolated by ultracentrifugation at 25 000 rpm (Beckmann, L8-70M) for 1.5 hours at 4°C. The pellet was removed and resuspended in DMEM, and lentivirus concentrations/titers were determined by quantitative RT-PCR using the Lenti-X qRT-PCR Titration kit (Clontech; catalog number 631235). Any residual plasmid DNA is removed by treatment with DNasel. The original virus preparation is immediately used for infection or divided into aliquots and stored at -80°C for future use.

Isolation of PBMCs.

Peripheral blood mononuclear cells (PBMCs) are prepared from whole blood or buffy coat. Whole blood is collected into 10 mL heparin vacuum tubes and either processed immediately or stored overnight at 4°C. Approximately 10 ml of anticoagulated whole blood is mixed with sterile phosphate-buffered saline (PBS) to obtain a total volume of 20 ml in a 50 ml conical centrifuge tube (PBS, pH 7.4, Ca ²⁺ /Mg ²⁺ free). 20 ml of this blood/PBS mixture is then gently applied to the surface of 15 ml of Ficoll-Paque® PLUS (GE Healthcare, 17-1440-03) before centrifugation at 400 g for 30-40 minutes at room temperature without braking. Buffy coat was purchased from Research Blood Components (Boston, MA). LeucoSep® tubes (Greiner bio-one) are prepared by adding 15 ml FicollPaque® (GE Health Care) and centrifuging at 1000 g for 1 minute. The buffy coat is diluted in a ratio of 1:3 in PBS (pH 7.4, without Ca ²⁺ or Mg ²⁺ ). The diluted buffy coat is transferred into a Leucosep tube and centrifuged at 1000 g for 15 minutes without braking. The layer of cells containing PBMCs observed at the diluted plasma/ficoll interface is carefully removed to minimize Ficoll contamination. Residual ficoll, platelets and plasma proteins are then removed by washing PBMCs three times with 40 ml PBS by centrifugation at 200 g for 10 minutes at room temperature. The cells are then counted using a hemocytometer. Washed PBMCs are washed once in CAR-T medium (AIM V-AlbuMAX® (BSA) (Life Technologies), 5% AB serum and 1.25 μg/ml amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U ./ml penicillin and 100 µg/ml streptomycin). Alternatively, washed PBMCs are transferred to insulated vials and frozen at -80°C for 24 hours before storing in liquid nitrogen for later use. T cell activation PBMC, derived from whole blood or buffy coat, are stimulated with magnetic beads conjugated to antibodies to human CD28 and CD3 for 24 hours before viral transduction. Freshly isolated PBMCs are washed once in CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies), 5% AB serum and 1.25 μg/ml amphotericin B (Gemini Bioproducts), 100 U/ml penicillin and 100 μg/ml streptomycin) without huIL-2 before resuspension at a final concentration of 1x106 cells/mg in CAR-T medium with 300 IU/ml human IL-2 (from a 1000x stock solution; Invitrogen). If PBMCs were previously frozen, they are thawed and resuspended at a concentration of 1x10 ⁷ cells/ml in 9 ml of prewarmed (37°C) cDMEM medium (Life Technologies) in the presence of 10% FBS, 100 units/ml penicillin and 100 μg ml streptomycin, at a concentration of 1x106 cells/ml before washing once in CAR-T medium, resuspending at a concentration of 1x106 cells/ml in CAR-T medium and adding IL-2 as described above.

Before activation, magnetic beads conjugated with antibodies to human CD28 and CD3 (available, for example, from Invitrogen, Life Technologies) are washed three times in 1 ml of sterile 1x PBS (pH 7.4), using a magnetic stand to release the beads from solution, before resuspended in CAR-T medium with 300 IU/ml human IL-2 to obtain a final concentration of 4x10 ⁷ beads/ml. PBMC and beads are then mixed in a 1:1 bead to cell ratio by transferring 25 μl (1x106 beads) of beads into 1 ml of PBMC. The desired number of aliquots are then dispensed into single wells of a 12-well low adherent or untreated cell culture plate and incubated at 37°C with 5% CO ₂ for 24 hours prior to viral transduction.

Transduction/transfection and expansion of T cells.

After incubation of PBMCs, cells are incubated for 48 hours at 37°C and 5% CO ₂ . The lentivirus is thawed on ice and 5x106 lentivirus is added along with 2 μl of TransPlus™ (Alstem) per ml of medium (final dilution 1:500) to each well containing 1x106 cells. Cells are incubated for an additional 24 hours before virus is added again. Alternatively, the lentivirus is thawed on ice and the corresponding virus is added at 5 or 50 MOI in the presence of 5 μg/ml polybrene (Sigma). Cells are centrifugally inoculated at 100g for 100 minutes at room temperature. The cells are then grown in the continuous presence of 300 IU/ml human IL-2 for 6-14 days (total incubation time depends on the final number of CAR-T cells required). Cell concentrations are analyzed every 2-3 days, at which time medium is added to maintain the cell suspension at a concentration of 1x106 cells/ml.

In some cases, activated PBMCs are electroporated using transcribed in

- 49 043737 vitro (IVT) RNA. In one embodiment, human PBMCs are stimulated with Dynabeads® (ThermoFisher) at a 1:1 ratio for 3 days in the presence of 300 IU/ml recombinant human IL-2 (R&D Systems) (other stimulating reagents may be used, such as TransAct T reagent Cell Reagent from Milyeni Pharmaceuticals). Before electroporation, the beads are removed. Cells are washed and resuspended in OPTI-MEM medium (ThermoFisher) at a concentration of 2.5 x 107 cells/ml. 200 µl of cell suspension (5x106 cells) is transferred into 2 mm Electroporation Cuvettes Plus™ (Harvard Apparatus BTX) and pre-cooled on ice. 10 μg of FVT TFP mRNA was added to the cell suspension. The mRNA/cell mixture was then electroporated at 200 V for 20 milliseconds using an ECM830 Electro Square Wave Porator (Harvard Apparatus BTX). Immediately after electroporation, cells are transferred to fresh cell culture medium (AIM V AlbuMAX (BSA) serum-free medium + 5% human serum AB + 300 IU/ml IL-2) and incubated at 37°C.

Verification of TFP expression by cell staining.

Following lentiviral transduction or electroporation of the mRNA, anti-mesothelin TFP expression is confirmed by flow cytometry using an anti-mouse Fab antibody to detect the mouse anti-mesothelin scFv. T cells are washed three times in 3 ml of staining buffer (PBS, 4% BSA) and resuspended in PBS at a concentration of 1x10 ⁶ cells per well. To eliminate dead cells, cells are incubated with LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen) for 30 minutes on ice. Cells are washed twice in PBS and resuspended in 50 μl of staining buffer. To block Fc receptors, 1 μl of a 1:100 dilution of normal goat IgG (BD Bioscience) was added to each tube and incubated on ice for 10 minutes. 1.0 ml FACS buffer is added to each tube, mixed well, and the cells are pelleted by centrifugation at 300 g for 5 minutes. Surface expression of scFv TFP is determined using R-phycoerythrin-labeled human MSLN IgG1 Fc Zenon® or human IgG1 isotype control. 1 μg of antibody is added to the appropriate samples and incubated for 30 minutes on ice. The cells are then washed twice and stained for surface markers using anti-CD3 APC (clone, UCHT1), Pacific blue anti-CD4 (clone RPA-T4), anti-CD8 APCy7 (clone SK1) from BD bioscience. Flow cytometry was performed using LSRFortessa™ X20 (BD Biosciences), data acquired using FACS diva software and analyzed using FlowJo® (Treestar, Inc. Ashland, OR).

Exemplary results are shown in FIG. 5A depicts surface expression analysis of activated PBMCs stained for CD8 (APCCy7 anti-CD8, y-axis) and mesothelin (MSLN) (R-phycoerythrin-labeled hMSLN IgG Zenon®, x-axis). Shown from left to right are cells that were either not transduced or transduced with the anti-MSLN-CD3ε, anti-MSLN-CD28ζ, and anti-MSLN-41BBζ; constructs. The proportion of CD8+, MSLN+ cells is shown in the upper right corner of each panel.

In fig. Figure 5B shows similar results from activated PBMCs stained for MSLN and ZPB that were transduced with TFP constructs containing native mesothelin single domain (SD) binding antibodies. Top row (left to right) shows untransduced cells as well as cells transduced with the positive control anti-MSLN-CD3ε TFP (SSI). Rows 2–4 show binding antibodies against MSLN SD1, SD4, and SD6, respectively, in cells transduced with (from left to right) CD3ε TFP, CD3yTFP, TCRe TFP, and CD28Z CAR constructs tagged with ZPB. The proportion of GFB+, MSLN+ cells is shown in the upper right corner of each panel.

Example 6: Cytotoxicity assay by flow cytometry.

Target cells that are mesothelin-positive or mesothelin-negative are labeled with the fluorescent dye carboxyfluorescein diacetate-I-succinimidyl ester (CFSE). These target cells are mixed with effector T cells that are either untransduced, transduced with control CAR-T constructs, or transduced with TFP. After the specified incubation time, the percentages of dead and live CFSE-labeled target cells and negative control target cells were determined for each effector/target cell culture by flow cytometry. The percentage of target cell survival in each T cell positive target cell culture is calculated relative to wells containing only target cells.

The cytotoxic activity of effector T cells is measured by comparing the number of surviving target cells without or with effector T cells after co-incubation of effector cells and target cells by flow cytometry. In experiments with anti-mesothelin TFP or CAR-T cells, the target cells are mesothelin-positive cells, while the cells used as a negative control are mesothelin-negative cells.

Target cells are washed once and resuspended in PBS at a concentration of 1x10 ⁶ cells

- 50 043737 current/ml. The fluorescent dye carboxyfluorescein diacetate-I-succinimidyl ester (CFSE) (ThermoFisher) was added to the cell suspension at a concentration of 0.03 μM, and the cells were incubated for 20 minutes at room temperature. The labeling reaction is stopped by adding complete cell culture medium (RPMI-1640 + 10% HI-FBS) to the cell suspension at 5 times the reaction volume, and the cells are incubated for an additional two minutes at room temperature. Cells are pelleted by centrifugation and resuspended in cytotoxic medium (RPMI1640 without phenol red (Invitrogen) plus 5% AB serum (Gemini Bioproducts) at a concentration of 2x105 cells/ml.

Fifty microliters of CFSE-labeled target cell suspension (equivalent to 10,000 cells) was added to each well of a 96-well U-bottom plate (Coming). Effector T cells transduced with anti-mesothelin TFP constructs, along with untransduced T cells as negative controls, are washed and suspended at a concentration of 2x106 cells/ml or 1x106 cells/ml in cytotoxic medium. 50 μl of effector T cell suspension (equivalent to 100,000 or 50,000 cells) is added to plated target cells to achieve an effector to target cell ratio of 10 to 1 or 5 to 1, respectively, for a total volume of 100 μl. The cultures are then mixed, centrifuged and incubated for four hours at 37°C and 5% CO ₂ . Immediately after incubation, 7AAD (7-aminoactinomycin D) (BioLegend) was added to the cultured cells as recommended by the manufacturer, and flow cytometry was performed using a BD LSRFortessa™ X-20 (BD Biosciences). Flow cytometry data analysis is performed using FlowJo® software (TreeStar, Inc.). The percentage of target cell survival is calculated by dividing the number of live target cells (CFSE+7-AAD-) in a sample with effector T cells and target cells by the number of live (CFSE+7-AAD-) cells in a sample with only cells- targets. The cytotoxicity of effector cells is calculated as the percentage of target cell destruction = 100% - percentage of cell survival.

T cells transduced with an anti-MSLN 28ζ CAR construct can demonstrate cytotoxicity toward mesothelin-expressing cells when compared with T cells that are either untransduced or transduced with a non-mesothelin-specific CAR control. However, T cells transduced with CD3ε against mesothelin can induce more effective target cytotoxicity than non-mesothelin CAR controls. CD3y anti-mesothelin TFPs may also mediate sustained cytotoxicity that would be greater than the cytotoxicity observed for anti-mesothelin CARs in effector to target cell ratios between 5 and 10:1. Some cytotoxicity may be observed with TCRa against mesothelin and TFP TCRe against mesothelin. Similar results can be obtained for anti-mesothelin TFPs designed with an alternative hinge region. Again, cytotoxicity to mesothelin-expressing target cells may be greater with TFP-transduced CD3ε anti-mesothelin or CD3y anti-mesothelin T cells than with CAR-transduced anti-mesothelin T cells. T cells electroporated with mRNAs encoding mesothelin-specific TFP may also exhibit robust cytotoxicity toward mesothelin-expressing cells. Because there is no significant killing of mesothelin-negative cells by control or TFP anti-mesothelin constructs, mesothelin-specific killing of mesothelin-expressing cells may be observed for T cells transduced with CD3ε anti-mesothelin CL or TFP anti-mesothelin CD3y CL.

Example 8: Determination of Cytotoxicity by Real-Time Cytotoxicity Assay TFPs against mesothelin can also demonstrate superior cytotoxicity compared to CARs against mesothelin in a real-time cytotoxicity assay (RTCA) format. The RTCA assay measures the electrical impedance of a monolayer of adherent target cells in each well in a specialized 96-well plate in real time and reports the final value recorded as a value called the cell index. Changes in the cellular index indicate disruption of the target cell monolayer as a result of the destruction of target cells by co-incubated T-cell effectors. Thus, the cytotoxicity of effector T cells can be assessed as the change in the cellular index in wells with both target cells and effector T cells compared to the cellular index in wells with only target cells.

Adherent target cells were cultured in DMEM, 10% FBS, 1% antibiotic-antifungal solution (Life Technologies). To prepare RTCA, 50 μl of, for example, DMEM media is added to the appropriate wells of an E-plate (ACEA Biosciences, Inc, Cat. No.: JL-10-156010-1A). The plate is then placed in the RTCA MP instrument (ACEA Biosciences, Inc.) at the appropriate plate position and the assay schedule is set in RTCA 2.0 software as described in the manufacturer's instructions. Baseline measurements are taken every 15 minutes for a total of 100 measurements. Then

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1 x 10 ⁴ target cells in a volume of 100 µl are added to each assay well and the T cells are allowed to settle for 15 minutes. The tablet is returned to the reader and readings continue.

The next day, effector T cells are washed and resuspended in cytotoxic medium (RPMI1640 without phenol red (Invitrogen) plus 5% AB serum (Gemini Bioproducts; 100318)). The plate is then removed from the instrument and effector T cells suspended in cytotoxic medium (RPMI1640 without phenol red + 5% AB serum) are added to each well at 100,000 cells or 50,000 cells to achieve an effector to target cell ratio of 10 to 1 or 5 to 1, respectively. The tablet is then placed back into the device. The measurement is performed every 2 minutes for a total of 100 measurements, and then every 15 minutes for a total of 1000 measurements. In the RTCA assay, killing of mesothelin-transduced cells can be observed by T cells transduced with CAR-transduced 28ζ T cells against mesothelin, as shown by the time-dependent decrease in cellular index after addition of effector cells compared to cells alone or cells co-incubated with T cells. transduced with a CAR control construct. However, the killing of target cells by TFP-expressing CD3ε anti-mesothelin T cells may be stronger and more rapid than that observed for anti-mesothelin CARs. For example, 4 hours after the addition of T cells transduced with TFP CD3ε against mesothelin, the killing of mesothelin-expressing target cells can be substantially complete. Little or no killing may be observed for T cells transduced with a number of TFP constructs containing other CD3 and TCR constructs. Similar results can be obtained for anti-mesothelin TFPs designed with an alternative hinge region. Cytotoxicity to mesothelin-transduced target cells may be greater with TFP-transduced CD3e anti-mesothelin or CD3y anti-mesothelin T cells than with CAR-transduced anti-mesothelin T cells.

The cytotoxic activity of TFP-transduced T cells may be dose dependent relative to the amount of virus (MOI) used for transduction. Increased killing of mesothelin-positive cells may be observed when the MOI of TFP CD3ε lentivirus against mesothelin is increased, further strengthening the relationship between TFP transduction and cytotoxic activity.

Exemplary results of the RTCA analysis are shown in FIG. 6. The anti-MSLN TFP construct was created by cloning the anti-MSLN scFv DNA fragment linked to the CD3ε DNA fragment via the DNA sequence encoding the linker: GGGGSGGGGSGGGGSLE (SEQ ID NO:1) into the p510 vector (from SBI) at the XbaI and EcoRI sites . The resulting anti-MSLN TFP construct was p510_antiMSLN_SS1_CD3ε (SS1 scFv anti-MSLN - linker - human CD3ε chain).

Full-length mesothelin (NM005823) was PCR amplified from pCMV6_XL4_Mesothelin (Origene) and cloned into XbaI and EcoRI restriction digested p527a (pCDH-EF1-MCS-T2A-Puro) (SBI) via Gibson recombination reaction. The target cells for RTCA were mesothelin-positive HeLa cells (cervical adenocarcinoma, ATCC® CCL2™), and mesothelin-negative PC-3 cells (prostate adenocarcinoma, ATCC® CRL1435™) were used as negative controls. Adherent target cells were cultured in DMEM with 10% FBS and 1% antibiotic-antifungal solution (Life Technologies).

A normalized cellular index indicating cytotoxicity was determined. Activated PBMCs were untreated (trace 1), untransduced (trace 2), or transduced with empty vector (trace 3), anti-MSLN TFP (anti-MSLN-CD3ζ TRuC, trace 4), anti-MSLN CAR with CD28Z signaling domain (trace 5 ) or 41BBZ (trace 6) (anti-MSLN-28ζ CAR and anti-MSLN-41BBζ CAR, respectively).

As shown in FIG. 6A, MSLN-positive HeLa target cells were efficiently killed by TFP-transduced anti-MSLN T cells compared to negative controls. In contrast, MSLN-negative PC-3 cells were not effectively killed by any of the constructs (Fig. 6B).

A similar experiment was performed using proprietary TFP constructs with single domain anti-mesothelin binding antibodies. In fig. 6C shows the killing of MSLN positive cells in a high target density cell line (HeLa-(MSLN ^hlgh )) using T cells obtained from two different human donors (top and bottom). Shown are cell killing tracks for TFP T cells with binding antibodies against MSLN SD1 (left), SD4 (middle), and SD6 (right). Activated PBMCs were untransduced (trace 1) or transduced with CD3ε TFP (trace 2), CD3y TFP (trace 3), TCRe TFP (trace 4), or CD28Z CAR (trace 5). The normalized cell index, indicating cytotoxicity, was established using a real time cell analyzer (RTCA) assay. As shown in the figure, all T cells except non-transduced ones were able to kill cancer cells.

Example 7: Luciferase-based cytotoxicity assay in cells with high or low target density.

The luciferase-based cytotoxicity assay (Luc-Cyto assay) evaluates the cytotoxicity of TFP-T and CAR-T cells by indirectly measuring luciferase enzyme activity in residual live target cells after coculture. The high target density cells used in the Luc-Cyto assay were HeLa-MSLN ^hlsh cells, and the low target density cells used were PC3 cells expressing low levels of mesothelin (PC3MSLN ^low ), all of which were stably transduced to express firefly luciferase. DNA encoding firefly luciferase was synthesized using GeneArt® (Thermo Fisher®) and inserted into multiple cloning sites of the single-promoter lentiviral vector pCDH527A-1 (System Bioscience). Firefly luciferase-carrying lentivirus was packaged as described above. HeLa-MSLN ^high or PC3-MSLN ^low cells were then transduced with the firefly luciferase construct carrying the lentivirus for 24 hours and then selected with puromycin (5 μg/ml). The production of HeLa-luc-MSLN ^high and PC3-luc-MSLN ^low luciferase cells was confirmed by measuring luciferase enzyme activity in the cells using the BrightGlo™ Luciferase Assay System (Promega).

Separate populations obtained from two different human donors were transduced with an empty expression vector (NT) or with the following TFP or CAR: anti-MSLN (positive control, SS1, 11 nM affinity), anti-MSLN-SDl (25 nM affinity) , anti-MSLN-SD4 (affinity 6 nM) or anti-MSLN SD6 (affinity 0.59 nM), each in the format CD3ε TFP, CD3y TFP, TCRe TFP, and CD28Z CAR. Two populations of transduced T cells were incubated with HeLa-MSLN ^high (Figure 7) or PC3-MSLN ^low (Figure 8).

Target cells were seeded at 5000 cells per well in a 96-well plate. TFP-T, CAR-T, or control cells were added to target cells in ratios of effector cells to target cells of 1:1 (black bars) or 1:5 (gray bars). The cell mixture was then cultured for 24 hours at 37°C with 5% CO2 before measuring luciferase enzyme activity in live target cells using the Bright-Glo® Luciferase Assay System. Cells were centrifuged to pellet and resuspended in medium containing luciferase substrate. Luciferase is released upon cell lysis, so high luciferase activity corresponds to a higher percentage of cell death.

Results using cells expressing high levels of MSLN are shown in FIG. 7. Shown is the % of cells killed in samples in the absence of T cells (target cells only), transduced with empty vector (NT), anti-MSLN (positive control, SS1) or with anti-mesothelin TFP-T cells with native anti-mesothelin binding antibodies. mesothelin SD1 (Figure 7A), SD4 (Figure 7B) and SD6 (Figure 7C), each in the format CD3ε TFP, CD3y TFP, TCRe TFP and CD28Z CAR. In each graph, black bars represent a 1:1 ratio of T cells to target cells and gray bars represent a 1:5 ratio of T cells to target cells. As can be seen in the figure, TFP-T cells, CAR-T cells, and the SS1 positive control all showed efficacy in killing MSLN. In fig. 8 shows a series of graphs demonstrating the activity of CAR-T cells against MSLN and TFP-T cells against a target cell line expressing low levels of mesothelin (PC3-MSLN ^low ). Shown is the % of cells killed in samples in the absence of T cells (target cells only), transduced with empty vector (NT), anti-MSLN (positive control, SS1), or with anti-mesothelin constructs SD1, SD4, and SD6 in TFP CD3ε formats (Figure 8A ), CD3y (Figure 8B), TCRe (Figure 8C), and CAR CD28Z (Figure 8D). In each graph, black bars represent a 1:1 ratio of T cells to target cells and gray bars represent a 1:5 ratio of T cells to target cells. Similar results were observed for the second T-cell donor.

As shown in FIG. , a 1:1 ratio of T cells to target cells resulted in the highest rate of target cell killing, as expected. In addition, TFP-T and CAR-T cells showed similar activity in cells expressing high levels of MSLN.

Example 8: Measurement of T cell activation by FACS.

Activation of T cells expressing anti-MSLN CAR and TFP constructs was performed using MSLN+ and MSLN- K562 cells as shown in FIG. 9. As described above, activated PBMCs were transduced with 50 MOI LV for two consecutive days and expanded. At day 8 post-transduction, PBMC co-cultures were plated with target cells (MSLN-overexpressing K562 cells) at a 1:1 E to T ratio (0.2 x 106 of each cell type) in cytotoxic medium (RPMI1640 without phenol red (Invitrogen) plus 5% serum AB (Gemini Bioproducts; 100318) K562 cells overexpressing BCMA were used as negative controls.24 hours after the start of coculture, cells were harvested, washed three times in PBS and stained with Live/Dead Aqua for 30 minutes on ice. To block Fc receptors, human Fc block (BD) was added and incubated for 10 minutes at room temperature.The cells were subsequently stained with anti-CD3 APC (clone, UCHT1), anti-CD8 APCy7 (clone SK1), Alexa Fluor® 700 CD69 (clone FN50) from BD Biosciences and PE against CD25 (clone BC96,

- 53 043737 eBioscience). Cells were washed twice and analyzed using BD LSRII-Fortessa. Data were analyzed as described above using FlowJo® analysis software (Tree star, Inc.). As shown in FIG. 9A, from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cells cocultured with MSLN − cells are shown in the top row, and cells cocultured with MSLN+ target cells are shown in the bottom row. The cells were then stained with antibodies specific to the surface activation markers CD69 and CD25. The number of cells stained with anti-CD69 antibodies corresponds to the x-axis, and the number of cells stained with anti-CD25 antibodies corresponds to the y-axis. As shown, T cells expressing anti-mesothelin CAR and TFP constructs were activated by culture with MSLN+ cells, as indicated by increased expression levels of CD69 and CD25, relative to coculture with MSLN− cells (Fig. 9B). The percentage of CD25+ cells for each construct in MSLN− (white bars) and MSLN+ (black bars) cells is shown.

A similar experiment was performed using MSLN- K562 cells (Figure 9C, circles) and MSLN+ K562 cells (Figure 9C, squares) for either untransduced T cells or T cells transduced with a positive control anti-MSLN binding antibody (510-SS1CD3ε). . Data represent the sum of CD25+, CD69+, and CD25+/CD69+ cells. In fig. 9D shows data for self-binding anti-MSLN antibodies SD1 (squares), SD4 (circles), and SD6 (triangles) in MSLN-targeted K562 cells (left panel) and MSLN+ K562 cells (right panel) combined with donor T cells. having TFP formats CD3ε, CD3y, TCRe and CD28Z CAR. Similar results were observed using cells from a second T cell donor.

T cell activation can be assessed in a similar manner by assaying granzyme B production. T cells are cultured and expanded as described above, and intracellular granzyme B staining is performed according to the manufacturer's kit instructions (Gemini Bioproducts; 100-318). Cells were collected, washed three times in PBS and blocked with human Fc block for 10 minutes. Cells were stained for surface antigens with anti-CD3 APC (clone UCHT1) and anti-CD8 APCy7 (clone SK1) for 30 minutes at 4°C. Cells were then fixed using a fixation/permeabilization solution (BD Cytofix/Cytoperm Fixation/Permealbilzation kit, P/N 554714) for 20 minutes at 4°C followed by a wash in BD Perm/Wash buffer. Cells were subsequently stained with Alexafluor700 against granzyme B (clone GB11), washed twice in BD Perm/Wash buffer, and resuspended in FACS buffer. Data were acquired using BD LSRII-Fortessa and analyzed using FlowJo® (Tree star Inc.).

As shown in FIG. 10A, from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cells cocultured with MSLN − cells are shown in the top row, and cells cocultured with MSLN+ target cells are shown in the bottom row. The number of cells stained with anti-GrB antibodies corresponds to the x-axis, and the number of cells stained with anti-CD8 antibodies corresponds to the y-axis. As shown, T cells expressing CAR and TFP constructs against mesothelin were activated by culturing with MSLN+ cells rather than with MSLN cells. These results are again shown in FIG. 10B, which shows the percentage of GrB+ cells for each construct in mesothelin-negative (MSLN-, white bars) and mesothelin-positive (MSLN+, black bars) cells. These data demonstrate the ability of MSLN-expressing cells to specifically activate T cells.

Example 9: Secretion of IL-2 and IFN-γ by ELISA.

Another dimension of effector T cell activation and proliferation associated with the recognition of cells bearing the recognition antigen is the production of effector cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ).

ELISA assays for human IL-2 (cat. no. EH2IL2, Thermo Scientific) and IFN-γ (cat. no. KHC4012, Invitrogen) were performed as described in the package inserts. In one example, 50 μl of reconstituted standards or samples is added in duplicate to each well of a 96-well plate, followed by the addition of 50 μl of biotinylated antibody reagent. The samples are mixed by gently tapping the plate several times. 50 µl of standard diluent is then added to all wells not containing standards or samples, and the plate is carefully closed with an adhesive plate cap before incubating for 3 hours at room temperature (20-25°C). The plate lid is then removed, the contents of the plate are removed, and each well is filled with wash buffer. This washing procedure is repeated only 3 times, and the tablet is blotted with paper towels or other absorbent material. Add 100 μl of the prepared streptavidin-HRP solution to each well and attach a new plate lid before incubating for 30 minutes at room temperature. The plate lid is removed again, the contents of the plate are removed and 100 µl of substrate solution is added to each well.

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TMB. The reaction is allowed to proceed at room temperature in the dark for 30 minutes, after which 100 μl of stop solution is added to each well. The tablet is being evaluated. Absorbance is measured using an ELISA plate reader set at 450 nm and 550 nm for 30 minutes until the reaction stops. The values at 550 nm are subtracted from the values at 450 nm and the amounts of IL-2 in the unknown samples are calculated relative to the values obtained using the IL-2 standard curve. Alternatively, perform 2-P1ex assays using the Human Cytokine Magnetic Buffer Reagent Kit (Invitrogen, LHB0001M) with the Human IL-2 Magnetic Bead Kit (Invitrogen, LHC0021M) and the Human IFN-γ Magnetic Bead Kit (Invitrogen, LHC4031M).

Briefly, 25 μl of human IL-2 and IFN-γ antibody beads are added to each well of a 96-well plate and washed using the following instructions: two washes in 200 μl of 1x wash solution, bring the plate into contact with a magnetic 96-well separator (Invitrogen, A14179 ), let the granules sit for 1 minute and decant the liquid. 50 μL of incubation buffer is then added to each well of the plate with 100 μL of reconstituted standards in duplicate or 50 μL of samples (supernatants from cytotoxicity assays) and 50 μL of dilution solution in triplicate, for a total volume of 150 μL. Samples are mixed in the dark at 600 rpm on an orbital shaker with an orbital radius of 3 mm for 2 hours at room temperature. The plate is washed following the same washing instructions, and 100 μl of biotinylated anti-human IL-2 and IFN-γ detection antibody is added to each well. Samples are mixed in the dark at 600 rpm on an orbital shaker with an orbital radius of 3 mm for 1 hour at room temperature. The plate is washed following the same washing instructions, and 100 μl of streptavidin-R-phycoerythrin is added to each well. Samples are mixed in the dark at 600 rpm. on an orbital shaker with an orbital radius of 3 mm for 30 minutes at room temperature. The plate is washed 3 times following the same washing instructions, and after decanting the liquid, the samples are resuspended in 150 μl of 1x wash solution. Samples are mixed at 600 rpm on an orbital shaker with an orbital radius of 3 mm for 3 minutes and stored overnight at 4°C. The plate is then washed following the same washing instructions and samples are resuspended in 150 µl of 1x wash solution.

The plate is read using MAGPIX System (Luminex) and xPONENT software. Data analysis is performed using MILLIPLEX Analyst software, which provides a standard curve and cytokine concentrations.

Relative to untransduced or control CAR-transduced T cells T cells transduced with anti-mesothelin TFP produce higher levels of IL2 and IFN-γ when co-cultured with cells that endogenously express mesothelin or mesothelin-transduced cells. In contrast, coculture with mesothelin-negative cells or non-transduced cells may result in little or no cytokine release from TFP-transduced T cells. Consistent with previous cytotoxicity data against mesothelin, TFPs engineered with an alternative hinge region can produce similar results when co-cultured with mesothelin-bearing target cells.

Consistent with previous cytotoxicity data, CD3ε against mesothelin and CD3y against mesothelin may result in the highest levels of IL-2 and IFN-γ in TFP constructs. However, cytokine production by T cells transduced with TFP CD3ε anti-mesothelin and TFP CD3y anti-mesothelin may be comparable to cytokine production by T cells expressing CAR 28ζ anti-mesothelin, despite TFPs exhibiting significantly higher levels of cell killing- targets. The possibility that TFPs may be more effective at killing target cells than CARs but release comparable or lower levels of proinflammatory cytokines represents a potential advantage of TFPs over CARs, as elevated levels of these cytokines are associated with dose-limiting toxicities of adoptive CAR-T therapy.

Exemplary results are shown in FIG. 11. As described above, activated PBMCs were transduced with 50 MOI of lentiviruses for two consecutive days and expanded. At day 8 post-transduction, PBMC co-cultures were plated with target cells (MSLN-overexpressing K562 cells) at a 1:1 E to T ratio (0.2 x 10 ^b of each cell type) in cytotoxic medium (RPMI1640 without phenol red (Invitrogen) plus 5% AB serum (Gemini Bioproducts; 100-318) K562 cells overexpressing BCMA were used as negative controls. After 24 hours, cells were analyzed for expression of IFN-γ (Figure 11A) and IL-2 (Figure 11B) by ELISA as described above.As shown in each figure from left to right, T cells were untransduced, empty vector transduced, transduced with anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cells co-cultured with MSLN cells are indicated by white bars, and cells co-cultured with MSLN+ target cells are indicated by black bars. As can be seen in Fig. 1, T cells expressing the anti-mesothelin CAR and TFP constructs were activated. as evidenced by the production as

IFN-γ and IL-2 by co-culture with MSLN+ cells rather than MSLN- cells, further demonstrating the ability of MSLN-expressing cells to specifically activate T cells.

Example 10: Exposure to CP107a by flow cytometry.

An additional assay for T cell activation is the surface expression of CD107a, a lysosomal membrane protein (also known as LAMP-1) that is located in the membrane of cytoplasmic cytolytic granules in quiescent cells. Degranulation of effector T cells, which is a prerequisite for cytolytic activity, leads to the mobilization of CD107a to the cell surface following activation-induced granule exocytosis. Thus, exposure to CD107a provides an additional dimension of T cell activation beyond cytokine production, which is closely correlated with cytotoxicity. Target and effector cells are separately washed and resuspended in cytotoxic medium (RPMI + 5% human serum AB + 1% antifungal solution with antibiotic). The assay is performed by combining 2x105 effector cells with 2x105 target cells in a 100 μl final volume in 96-well U-bottom plates (Corning) in the presence of 0.5 μl/well PE/Cy7 labeled anti-human CD107a antibody (LAMP-1) ( clone H4A3, BD Biosciences). The cultures are then incubated for one hour at 37°C and 5% CO ₂ . Immediately after this incubation, 10 μl of a 1:10 dilution of monensin secretion inhibitor (1000x solution, BD GolgiStop™) was carefully added to each well without shaking the cells. The plates are then incubated for an additional 2.5 hours at 37°C and 5% CO2. After this incubation, cells are stained with anti-human CD3 APC (clone UCHT1, BD Biosciences), anti-human CD8 PerCP/Cy5.5 (clone SKI, BD Biosciences) and anti-human CD4 Pacific Blue (clone RPA-T4, BD Biosciences) and then incubated for 30 minutes at 37°C and 5% CO2. Cells are then washed 2 times in FACS buffer (and resuspended in 100 μl FACS buffer and 100 μl IC fixation buffer before analysis.

The effect of CD107a on the surface of T cells was determined by flow cytometry. Flow cytometry was performed using LSRFortessa™ X20 (BD Biosciences), and flow cytometry data analysis was performed using FlowJo software (Treestar, Inc. Ashland, OR). The percentage of CD8+ effector cells within the CD3 gate that are CD107 positive is determined for each effector/target cell culture.

Consistent with previous cytotoxicity and cytokine data, coculture of mesothelin-expressing target cells with effector T cells transduced with CAR 28ζ against mesothelin can induce increased surface expression of CD107a relative to effectors incubated with mesothelin-negative cells. targets. In comparison, under the same conditions, TFP-expressing effectors CD3ε anti-mesothelin DL or CD3y anti-mesothelin DL can demonstrate a 5- to 7-fold induction of CD107a expression. Anti-mesothelin TFPs engineered with an alternative hinge region can produce similar results when co-cultured with mesothelin-bearing target cells.

Example 11: In vivo efficacy studies in mice.

To evaluate the ability of effector T cells transduced with TFP against mesothelin to achieve antitumor responses in vivo, effector T cells transduced with CAR 28ζ against mesothelin, TFP DL CD3ε against mesothelin, or TFP DL CD3y against mesothelin were adoptively transferred into NOD/SCID mice. /IL-2Ry-/- (NSG-JAX), which were previously inoculated with human mesothelin+ cancer cell lines.

Female NOD/SCID/IL-2Ry-/- (NSG-JAX) mice, at least 6 weeks old at study entry, were obtained from The Jackson Laboratory (source number 005557) and acclimatized for 3 days before use in the study. . Human mesothelin-expressing cell lines for inoculation are maintained in log-phase culture before they are harvested and counted using trypan blue to determine the number of viable cells. On the day of the tumor test, cells are centrifuged at 300 g for 5 minutes and resuspended in prewarmed sterile PBS at a cell concentration of 0.5-1x106 cells/100 μl. For adoptive transfer, T cells are prepared either untransduced or transduced with CAR 28ζ anti-mesothelin, TFP DL CD3ε anti-mesothelin, or TFP DL CD3y anti-mesothelin constructs. On day 0 of the study, 10 animals per experimental group were intravenously injected with 0.5-1x106 mesothelin-expressing cells. After 3 days, each animal is intravenously transferred to 5x106 effector T cell populations in 100 μl of sterile PBS. Detailed clinical observations on animals are recorded daily until euthanasia. Body weight is measured for all animals weekly until death or euthanasia. All animals are euthanized after 35

- 56 043737 days after the adoptive transfer of the study and control drugs. All animals that become distressed during the study are sacrificed at the discretion of the study director in consultation with a veterinarian.

Compared with untransduced T cells, adoptive transfer of T cells transduced with CAR 28ζ anti-mesothelin, TFP DL CD3ε anti-mesothelin, or TFP DL CD3y anti-mesothelin may improve the survival of mice harboring a mesothelin-expressing tumor cell line and may indicate that that T cells transduced with anti-mesothelin CAR and TFP constructs are able to mediate target cell killing, correspondingly enhancing survival in these mouse models. Taken together, these data may indicate that TFPs are an alternative platform for the generation of chimeric receptors that exhibit superior antigen-specific killing compared to first-generation CARs both in vitro and in vivo.

Example 12: Treatment with human T-cells TFP in a solid tumor xenograft mouse model in vivo.

The efficacy of human TFP.mesothelin T cell treatments can also be tested in immunocompromised mouse models harboring subcutaneous solid tumors derived from human mesothelin-expressing ALL, CLL, NHL, or MSTO cell lines. Tumor shrinkage in response to treatment with human TFP.mesothelin T cells can be assessed either by measuring tumor size with a caliper or by monitoring the intensity of the green fluorescent protein (GFP) signal emitted by GFP-expressing tumor cells.

Primary human solid tumor cells can be grown in immunocompromised mice without the need for in vitro culture. Examples of solid cancer cells include solid tumor cell lines such as those proposed in The Cancer Genome Atlas (TCGA) and/or the Broad Cancer Cell Line Encyclopedia (CCLE, see Barretina et al ., Nature 483:603 (2012). Examples of solid cancer cells include primary tumor cells isolated from mesothelioma, renal cell carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, cancer liver, pancreatic cancer, kidney, endometrial and stomach cancer. In some ways of carrying out cancer to be treated, they are selected from a group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, ovarian light cell cancer, mixed ovarian carcinomas, endometrhyoid ovarian mucosa, pancreatic adenocarcinoma, prototal adenocarcycia gastric gland, serous carcinoma of the uterus, adenocarcinoma lung, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma and mammary adenocarcinoma. These mice can be used to test the effectiveness of TFP.mesothelin T cells in xenograft human tumor models (see, for example, Morton et al., Nat. Procol. 2:247 (2007)). After subcutaneous implantation or injection of 1x106-1x107 primary cells (collagenase-treated bulk tumor suspensions in EC matrix material) or tumor fragments (primary tumor fragments in EC matrix material), tumors are allowed to grow to a volume of 200-500 mm ³ before treatment.

One such experiment was conducted to test the in vivo efficacy of MSLN-specific single domain antibodies (sdAbs) in xenograft mouse models of mesothelioma, as described above. Luciferase-labeled MSTO-211H-FL-MSLN-Luc) were inoculated subcutaneously at a concentration of 1x106 cells per mouse with Matrigel® in a 1:1 ratio. Tumor volume was monitored by measuring with a caliper twice a week. Fourteen days after tumor injection, when the tumor volume was approximately 300 mm ³ , each animal was injected intravenously with 1 x 10 ⁷ T cells. T cells used included cells transduced with CD3ε-SD1 TFP, CD3y-SD1 TFP, CD3ε-SD4 TFP, CD3y-SD4 TFP, CD28Z SD1 CAR, and CD28Z SD1 CAR. A group of mice that did not receive T cell injection was used as a negative control.

The results are shown in Fig. 12A. Mice injected with CD3ε-SD1 TFP and CD3y-SD1 TFP T cells showed the largest and most rapid reduction in tumor volume, although mice not injected with T cell control showed a reduction in tumor volume after T cell injection.

The sustained efficacy of SD1ε and y-TFP T cells was tested in vivo through a repeat study using surviving mice in a xenograft mouse model of mesothelioma.

Mice were inoculated subcutaneously with 1x106 tumor cells (MSTO 211H FL MSLN Luc) per mouse with Matrigel® (1 to 1 ratio). One group of mice was injected with Raji cells as a negative control, and one group of mice was injected with MSTO cells only, again as a negative control. Tumor volume was monitored by measuring with a caliper twice a week. Fourteen days after tumor injection (when the tumor volume had reached approximately 300 mm ³ ), each animal was intravenously injected with 1x10 ⁷ MSTO (MSLN+) or Raji (MSLN-, as a negative control). The results are shown in Fig. 12V. Each line on the figure represents one animal. As shown in FIG. , mice that had previously received anti-MSLN TFP-T cell treatment were capable of recurrent tumor volume reduction or tumor eradication, indicating that the mice either retained the originally administered T cells or developed a secondary anti-MSLN response . In contrast, mice reintroduced with Raji cells (MSLN−) were unable to control the growth of Raji tumors, thus demonstrating the specificity of the TFP-T cell response.

Example 13: In Vivo Efficacy of MSLN ε-TFP-T Cells Derived from Patients in a Xenograft Mouse Tumor Model MSLN SD1 ε-TFP-T Cells Derived from Patients with Ovarian Cancer were used to test antitumor efficacy in vitro and in vivo SD1 ε-TFP-T cells against mesothelin-expressing tumor cells (MSTO-MSLN-Luc). Lentivirus was prepared as described above. Preparation of CD4+ and CD8+ T Cells from Whole Blood of Ovarian Cancer Patients CD4 and CD8 T cells were purified from whole blood of ovarian cancer patients as follows (schematic overview shown in FIG. 13A). 40-50 ml of heparinized whole blood from patients with ovarian cancer was collected and transported overnight using Conversant Bio (Huntsville, AL). Blood was diluted with an equal volume of PBS, and 35 ml of diluted whole blood was carefully applied to 15 ml of Ficoll-Paque® (GE healthcare, Cat. No: 17-5442-02) in a 50 ml conical tube. It was then centrifuged at 800 x g for 20 minutes at room temperature in a swing swing rotor without braking. The top layer was aspirated, leaving the layer of mononuclear cells (lymphocytes, monocytes and platelets) undisturbed in interphase. The mononuclear cell layer was transferred to a new 50 ml conical tube, 30 ml PBS was added and centrifuged at 300xg for 10 minutes at room temperature. Add 1-2 ml of lysis buffer ACK (ThermoFisher, cat. no.: A1049201) to the sediment, mix thoroughly and incubate at room temperature for 2 minutes, add 20 ml of PBS, centrifuge at 300xg for 10 minutes at room temperature. The cell pellet was resuspended in 10 ml of ice-cold MACs buffer and cells were counted using Cellometer Auto 2000. Isolation of CD4+ and CD8+ T cells was performed using human CD4/8 Miltenyi microbeads (cat. no. 130-045-101; 130045-201) in accordance with the manufacturer's instructions.

TFP T cells were prepared as described above, and transduction was determined by FACS. Mesothelin expression was confirmed on target cells (MSLN ^hlgh cell line MSTO211H-FL MSLN (derived from its own parent MSTO-211H, ATCC, CRL-2081)), and MSLN-Fc expression was confirmed on SD1 ε-TFP T cells by flow cytometry in the same day as the luciferase assay. One suspension of luciferase-labeled target cells (MSTO-211H-FL MSLNLuc or MSLN cell line SZO-Luc (A2780, Sigma)) was prepared in R10 medium. 1 x 10 ⁴ target cells in 100 μl were added to a 96-well flat-bottom plate. TFP-T cells were added to 100 μl at a different ratio of effector cells to target cells (E:T) as indicated. Determination of FACS-based transduction efficiency and T cell activation

TFP-T cells were thawed, degranulated (expanded ex vivo under Dynabeads+IL-2 conditions), washed, and then resuspended in cytokine-free T cell culture medium. The desired number of T cells (in 100 μl) was added to achieve an effector cell to target cell ratio of 5 to 1, 1 to 1, and 1 to 5, respectively. For each type of T cells, three replicates were prepared at the tested ratio. The cells were then cultured for 24 hours at 37°C and 5% CO2. After 24 hours of co-culture, the plate was centrifuged at 300 x g for 2 minutes to pellet the cells. 100 μL of culture supernatant from each well was carefully removed for Luminex analysis. 100 ml of assay buffer from the Bright-Glo™ Luciferase Assay System (Promega, #E2650) was added to each well. The contents in each well were mixed by gently pipetting up and down. The mixture of cells and reagent was left at room temperature in the dark for 3 minutes to completely lyse the cells. 200 μl of cell lysate from each well was transferred to a 96-well Greiner-One white-walled plate. Luminescence was measured in relative luminescence units (RLU) using an M5 SpectraMax plate reader (Molecular devices).

The percentage (%) of tumor lysis was calculated using the formula below: Luminescence (Tumor + T cell) % tumor lysis = 100* [1 - -------------------- -----------------]

Luminescence (Tumor)

Luminex® analysis.

Tumor-T cell co-culture supernatant was collected and stored at −80°C as previously described. Cytokine profiles were determined using a Millipore Luminex kit (HCD8MAG-15K) according to the manufacturer's instructions. Supernatant liquid

- 58 043737 was applied to the plate without dilution and measured using Magpix xMAP® technology.

Xenograft Mouse Model of Subcutaneous Mesothelioma and In Vivo Assessments 6-week-old female NSG mice (NOD.Cg-Prkdc ^scid Il2rg ^tmlW 'l/SzJ, Cat#: 005557, Jackson Laboratories) were used in this study. Animals were acclimatized for at least 3 days under conditions similar to those of the study. MSTO-211H-FLMSLN-Luc cells were suspended in sterile PBS at a concentration of 1x106 cells/100 µl. The cell suspension in PBS was then mixed 1:1 with ice-cold Matrigel® to produce a final injection volume of 200 μl for each mouse. The resulting cell suspension in PBS/Matrigel® was kept on ice until subcutaneously injected into the hindquarters of the mouse. Tumor growth was monitored as tumor volume by measuring with a caliper. Tumor volume was calculated as follows:

Ten days after tumor cell injection, animals were randomized according to tumor volume (200~300 mm ³ ) and divided into 10 groups to receive injection of SD1 ε-TFP-T cells from different patients (the number of mice per group varies depending on the number of SD1 ε-TFPT cells recovered on the day of injection). The day the T-cell injection was performed was considered day 0 of the study. T cells were prepared in sterile PBS at a concentration of 5x106 cells/100 μl. The cell suspension was then injected intravenously into the tail vein of the mice.

Ex vivo expansion of SD1 E-TFP-T cells derived from ovarian cancer patients MSLN-specific sdAb TFP-T cells were prepared using lentiviral CD3ε-encoding TFP formats with SD1 binding antibodies targeting MSLN. Fold expansion determined by viable cell counts on day 10 ranged from 8.58 to 28.2 times (17.8 +/- 3.3) compared to day 0 for cells obtained with Dynabeads®+IL- 2, and from 10 to 33.6 times (22.9 +/5.0) compared with day 0 for cells obtained with TransAct® + IL-7/15. Transduction efficiency for SD1 ε-TFP-T cells was determined at day 10 of expansion by surface staining for the presence of ZPB and MSLN-Fc on CD4+ and CD8+ populations. Transduction efficiency ranged from 28.6% to 52.1% (40.9 +/- 4.0%) for Dynabeads+IL-2-derived cells and from 5.7% to 46.9% (26.8 +/- 6.3%) for cells obtained with TransAct+IL-7/15; no significant differences in fold expansion and transduction efficiency were observed between Dynabeads+IL-2 and TransAct+IL-7/15 conditions. The number of vector copies per cell corresponded to the transduction efficiency, with the copy number being about 1~2 per cell under Dynabeads+IL-2 or TransAct+IL-7/15 conditions, with the exception of patient 1, who had 0.38 vector copies per cell. cell.

In vitro antitumor activity of SD1 ε-TFP T cells derived from ovarian cancer patients

The in vitro efficacy of SD1 ε-TFP-T cells derived from ovarian cancer patients was tested using luciferase-based reporter tumor cell lysis assays. Mesothelin expression was confirmed in MSTO-211H-FLMSLN-Luc cell lines on the day of analysis (Figure 13B); all SD1 ε-TFP T cells demonstrated varying levels of tumor killing. Sustained tumor cell lysis was observed in patients 1, 2, 4 and 5 (75%~97%). MSLN ε-TFP-T cells co-cultured with MSTO-211H-FLMSLN-Luc (MSLN high expressor) at a 5 to 1 effector cell to target cell ratio, with Patient 3 demonstrating ~35% tumor lysis at an effector cell to cell ratio -targets 5 to 1, 4 of 5 patients (patients 1, 2, 4 and 5) demonstrated an average of 50% tumor lysis with a ratio of effector cells to target cells of 1 to 1, 2 of 5 (patients 4 and 5) demonstrated ~ 50% tumor lysis even with a ratio of effector cells to target cells of 1 to 5. All T cells demonstrated rapid destruction of tumor cells. Tumor lysis was not observed for all MSLN ε-TFP™ T cells when co-cultured with mesothelin-negative S3O-Luc cell lines (Figure 13C). MSLN ε-TFP cytokine profiles of five patients were analyzed using the Luminex® human CD8 panel, cytolytic cytokines such as IFN-γ, GM-CSF, granzyme-A/B, IL-2, MIP1α/β, TNF-α, and perforin were significantly increased in MSLN ε-TFP™ T cells compared to non-transduced T cells (Figure 13D-L).

The in vivo efficacy of MSLN ε-TFP-T cells in a xenograft mouse model of an MSLN-expressing tumor, MSTO-211H-FLMSLN-Luc, was used to establish a mouse model of a subcutaneous xenograft mesothelin-expressing tumor. Tumor volume was measured twice a week. On day 10 after tumor injection, the average tumor volume reached 200-300 mm ³ , and MSLN ε-TFP T cells expanded for 10 days from one normal donor (ND12, Fig. 14A) and patients 1-4 (Fig. 14B-E) were thawed and the efficiency of transduction was confirmed. 5x106 MSLN ε-TFP T cells or non-transduced T cells per mouse were injected i.v., and tumor volumes were monitored. MSLN ε-TFP-T cells from 3 of 4 patients (patients 1, 2, and 4) demonstrated complete tumor clearance by day 20 after T cell injection. Tumor removal was maintained

- 59 043737 until 40 days. Five of six mice received MSLN ε-TFP-T cells from patient 3, which showed partial protection. Of all four patients who received MSLN ε-TFP-T cells from ND12, one demonstrated complete tumor clearance and two patients demonstrated partial tumor clearance.

Notes.

Although preferred embodiments of the present invention have been shown and described herein, those skilled in the art will appreciate that these embodiments are provided for purposes of illustration only. Many variations, changes and substitutions will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. It should be understood that various alternative embodiments of the present invention described herein may be used in the practice of the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of this claim, as well as their equivalents, will be covered by this claim.

Appendix A: Summary of Sequences

SEQID NO. Name Subsequence 1 Short linker 1 GGGGSGGGGSGGGGSLE 2 Short linker 2 AAAGGGGSGGGGSGGGGSLE 3 Long linker AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE 4 Human CD3-S MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGIT QTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIG GDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPR GSKPEDANFYLYLRARVCENCMEMDVMSVATIVI VDICITGGLLLLVYYWSKNRKAKAKPVTRGAGA GGRQRGQNKERPPPVPNPDYEPIRKGQRD LYSGL NQRRI 5 Human CD3-y MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYD YQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDK KKWNLGSNAKDPRGMYQCKGS QNKSKPLQVYY RMCQNCIELNAATISGFFLFAEIVSIFVLAVGVYFIA GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQ YSHLQGN QLRRN 6 Human CD3-5 MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVN CNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYR CNGTDIYKDKESTVQVHYRMCQSCVELDPATVA GIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQ ALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS 7 POP-ζ human MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYL LDGILFIYGVILTALFLRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR 57 Human TCR α chain MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIML LVDGKQQMVVVCLVLDVAPPGLDSPIWFSAGNG SALDAFTYGPSPATDGTWTNLAHLSLPSEELASW EPLVCHTGPGAEGHSRSTQPMHLSGEASTARTCP QEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSC LCDPAGPLPSPATTTRLRALGSHRLHPATETGGRE ATS SPRPQPR DRRWGDTPPGRKPGSPVWGEGSYL

- 60 043737

SSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPP LQAGA 9 TCR α chain C region person PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV SQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKS FETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS 10 Human TCR α chain V region CTL-L17 MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQ NSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPA EGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIV PSQPGDSAVYFCAAKGAGTASKLTFGTGTRLQVT L eleven TCR β-chain C region person EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATG FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSV SYQQGVLSATILYEILLGKATLYAVLVSALVLMA MVKRKDF 12 Human TCR β-chain V region CTL-L17 MGTSLLCWMALCLLGADHADTGVSQNPRHNITK RGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTY FQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQ GDSAMYLCASSLAGLNQPQHFGDGTRLSIL 13 Human TCR β-chain V region YT35 MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEM GQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYF NNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEP RDSAVYFCASSFSTCSANYGYTFGSGTRLTVV 14 DNA sequence MSLN acgcgtgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagtt agcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtgga agtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacat ggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcct agctcgatacaataaacgggtctctctggttagaccagatctgagcctgggag ctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgag tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctc agacccttttagtcagtgtggaa aatctctagcagtggcgcccgaacagggac ctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctg aagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaa aattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagt attaagcgggggagaatt agatcgcgatgggaaaaaattcggttaaggccag

- 61 043737

ggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagct agaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagaca aatactgggacagctacaaccatcccttcagacaggatcagaagaacttagat cattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaa agacaccaag gaagcttttagacaagatagaggaagagcaaaacaaaagtaa gaccaccgcacagcaagcggccactgatcttcagacctggaggaggagata tgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaac cattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaa aaagagcagtgggaataggagctttgttcc ttgggttcttgggagcagcagga agcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattatt gtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaac agcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatc ctggctgtggaaagatacctaaaggat caacagctcctggggatttggggttg ctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaat aaatctctggaacagattggaatcacacgacctggatggagtgggacagaga aattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccag caagaaaagaatgaacaagaattattggaatt agataaatgggcaagtttgtgg aattggtttaacataacaaattggctgtggtatataaaattattcataatgatagta ggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagtt aggcagggatattcaccattatcgtttcagacccacctcccaaccccgagggg acccgacaggcccgaaggaatagaagaaga aggtggagagagagacaga gacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaa gaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataa tagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaa ttttatcgatactagtattatgcccagtacatg accttatgggactttcctacttggc agtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac atcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc attgacgtcaatggga gtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgc cccattgacgcaaatgggcggtaggcgtgtacggtgggaggtttatataagca gagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttg acctccatagaagattctagagccgccaccat gcttctcctggtgacaagcctt ctgctctgtgagttaccacacccagcattcctcctgatcccagacattcagcag gtccagctccagcagtctggccctgaactcgaaaaacctggcgctagcgtga aaatttcctgtaaagcctccggctactcttttactggctacacaatgaattgggtg aaacagtctcacggcaa atccctcgaatggatcggactcatcacaccctacaat ggcgcctcttcctacaaccagaaattccggggcaaggcaacactcactgtgg acaaatcatcctctaccgcctacatggatctgctctccctcacatctgaggactc

- 62 043737

cgctgtctacttttgtgcccgaggaggatacgacggacgaggattcgattactg gggacagggaacaactgtgaccgtgtctagtggcggcggagggagtggag gcggaggatcttctggcgggggatccgatattgaactcacacagtctcccgct atcatgtctgcttctcccggcgagaaagtgactatgacttgctct gcttcctcttct gtgtcctacatgcactggtaccagcagaaatctggcacatcccctaaacggtg gatctacgatactagcaaactggcatccggcgtgcctgggcgattctctggctc tggctctggcaactcttactctctcacaatctcatctgtcgaggctgaggacgat gccacatactactgtcagcagtggtctaaacacccactcacattc ggcgctgg cactaaactggaaataaaagcggccgcaggtggcggcggttctggtggcgg cggttctggtggcggcggttctctcgaggatggtaatgaagaaatgggtggtat tacacagacaccatataaagtctccatctctggaaccacagtaatattgacatgc cctcagtatcctggatctgaaatactatggcaacacaatgataa aaacataggc ggtgatgaggatgataaaaacataggcagtgatgaggatcacctgtcactgaa ggaattttcagaattggagcaaagtggttattatgtctgctaccccagaggaag caaaccagaagatgcgaacttttatctctctacctgagggcaagagtgtgtgaga actgcatggagatggatgtgatgtcggtggccacaattgt catagtggacatct gcatcactgggggcttgctgctgctggtttactactggagcaagaatagaaag gccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaag gggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcc catccggaaaggccagcgggacctgtattctggcctgaatcagagacg catct gataagaattcgatccgcggccgcgaaggatctgcgatcgctccggtgcccg tcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggag gggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactggg aaagtgatgtcgtgtactggctccgcctttttcccga gggtgggggagaaccgt atataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccag aacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgc cctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgc ctgtggt gcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtc gagaccgggcctttgtccggcgctcccttggagcctacctagactcagccgg ctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgt tctgcgccgttacagatccaagctgtgaccgg cgcctacgctagatgaccgag tacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgta cgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtc gatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctca cgcgcgtcgggctcga catcggcaaggtgtgggtcgcggacgacggcgcc gcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgtt cgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgc gcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccg

- 63 043737

cgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtct gggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggg gtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcg gctcggcttcaccgtcaccgccgacgtcga ggtgcccgaaggaccgcgcac ctggtgcatgacccgcaagcccggtgcctgagtcgacaatcaacctctggatt acaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatg tggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattt tctcc tccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgt caggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggtt ggggcattgccaccacctgtcagctccttccgggactttcgctttccccctccc tattgccacggcggaactcatcgcc gcctgccttgcccgctgctggacaggg gctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcc tttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctg ctacgtcccttcggccctcaatccagcggaccttccttcccgcgg cctgctgcc ggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctc cctttgggccgcctccccgcctggtacctttaagaccaatgacttacaaggcag ctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattc actcccaacgaaaataagatctgctttt tgcttgtactgggtctctctggttagac cagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgact ctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag tagtagtt catgtcatcttattattcagtatttataacttgcaaagaaatgaatatca gagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaata gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtc caaactcatcaatgtatcttatcatgtctggctctagctatcccgcccc taactcc gcccagttccgcccattctccgccccatggctgactaatttttttatttatgcaga ggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctagacttttgcagagacggcccaaattcgtaatcatggtcatagct gtttcctgtgtgaaattgttatccgctcacaatt ccacacaacatacgagccgga agcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaatt gcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcat taatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttc cgcttcctc gctcactgactcgctgcgctcggtcgttcggctgcggcgagcgg tatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataac gcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaa aaaggccgcgttgctggcgtttttccataggctccgcccccctgac gagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaa gataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccc

- 64 043737

tgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttc tcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaa ctatcgtcttgagtccaacc cggtaagacacgacttatcgccactggcagcag ccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttc ttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgc gctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggc aaacaaaccaccg ctggtagcggtggtttttttgtttgcaagcagcagattacgc gcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacg ctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa ggatcttcacctagatccttttaaattaaaaatgaagtt ttaaatcaatctaaagtat atatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagata actacgatacgggaggcttaccatctggccccagtgctgcaatgataccgcg agaccacgctcaccggctccagatt tatcagcaataaaccagccagccgga agggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatt aattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaac gttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttc at tcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtg caaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggc cgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgc catccgtaagatgcttttctgtgactggtga gtactcaaccaagtcattctgagaa tagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatacc gcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcgggg cgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc gtgcacccaactga tcttcagcatcttttactttcaccagcgtttctgggtgagca aaaacaggaaggcaaaatgccgcaaaaaaagggaataagggcgacacggaa atgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattg tctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatagg ggttc cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcat gacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttc ggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacag cttgtctgtaagcggatgccggggag cagacaagcccgtcagggcgcgtcag cggggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagatt gtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaagga gaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaa gggcgatcgg tgcgggcctcttcgctattacgccagctggcgaaagggggat

- 65 043737

gtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgtt gtaaaacgacggccagtgccaagctg 15 amino acid sequence MSLN: human mesothelin sequence (UniProt accession number Q13421) MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSR TLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFP CAEVSGLSTERVRELAVALAQKNVKLSTEQLRCL AHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTR FFSRITKANVDLLPRGAPERQRLLPAALACWGVR GSLLSEADVRALGGLACDLPGRFVAESAEVLLPR LVSCPGPLD QDQQEAARAALQGGGPPYGPPSTWS VSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSS RDPSWRQPERTILRPRFRREVEKTACPSGKKAREI DESLIFYKKWELEACVDAALLATQMDRVNAIPFT YEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKM SPEDIRKWNVTSLETLKALLEVNKGHEMSPQ VAT LIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEE LSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLA FQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVS MDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGL KAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPN GYLVLDLSMQEALSGTPCLLGPG PVLTVLALLA STLA 16 DNA p510_anti- MSLN_SSl_CD3 _e acgcgtgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagtt agcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtgga agtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacat ggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcct agctcgatacaataaacgggtctctctggttagaccagatctgagcctgggag ctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgag tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctc agacccttttagtcagtgtggaa aatctctagcagtggcgcccgaacagggac ctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctg aagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaa aattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagt attaagcgggggagaatt agatcgcgatgggaaaaaattcggttaaggccag ggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagct agaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagaca aatactgggacagctacaaccatcccttcagacaggatcagaagaacttagat cattatataatacagtagcaaccctct attgtgtgcatcaaaggatagagataaa

- 66 043737

agacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaa gaccaccgcacagcaagcggccactgatcttcagacctggaggaggagata tgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaac cattaggagtagcacccaccaaggcaaagagaagagtggtgcagagaagaaa aaagagcagtgggaataggagct ttgttccttgggttcttgggagcagcagga agcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattatt gtctggtatagtgcagcagcagaacaatttgctgaggctattgaggcgcaac agcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatc ctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttg ctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaat aaatctctggaacagattggaatcacacgacctggatggagtgggacagaga aattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccag caagaaaagaatgaaca agaattattggaattagataaatgggcaagtttgtgg aattggtttaacataacaaattggctgtggtatataaaattattcataatgatagta ggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagtt aggcagggatattcaccattatcgtttcagacccacctcccaaccccgagggg acccgacaggcccga aggaatagaagaagaaggtggagagagagacaga gacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaa gaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataa tagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaa ttttatcgatactagtattatg cccagtacatgaccttatgggactttcctacttggc agtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac atcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccc attgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaat g tcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtg ggaggtttatataagcagagctcgtttagtgaaccgtcagatcgcctggagac gccatccacgctgttttgacctccatagaagattctagagccgccaccatgcttc tcctggtgacaagccttctgctctgtgagttaccacaccca gcattcctcctgat cccagacattcagcaggtccagctccagcagtctggccctgaactcgaaaaa cctggcgctagcgtgaaaatttcctgtaaagcctccggctactcttttactggcta cacaatgaattgggtgaaacagtctcacggcaaatccctcgaatggatcggac tcatcacaccctacaatggcgcctcttcctaca accagaaattccggggcaag gcaacactcactgtggacaaatcatcctctaccgcctacatggatctgctctccc tcacatctgaggactccgctgtctacttttgtgcccgaggaggatacgacggac gaggattcgattactggggacagggaacaactgtgaccgtgtctagtggcgg cggagggagtggaggcggaggatcttctggcgggg gatccgatattgaactc acacagtctcccgctatcatgtctgcttctcccggcgagaaagtgactatgactt gctctgcttcctcttctgtgtcctacatgcactggtaccagcagaaatctggcac

- 67 043737

atcccctaaacggtggatctacgatactagcaaactggcatccggcgtgcctg ggcgattctctggctctggctctggcaactcttactctctcacaatctcatctgtc gaggctgaggacgatgccacatactactgtcagcagtggtctaaacacccact cacattcggcgctggcactaaactggaaataaaagcggccgcaggtggcgg c ggttctggtggcggcggttctggtggcggcggttctctcgaggatggtaatg aagaaatgggtggtattacacagacaccataaagtctccatctctggaacca cagtaatattgacatgccctcagtatcctggatctgaaatactatggcaacacaa tgataaaaacataggcggtgatgaggatgataaaaacataggcagtgatga g gatcacctgtcactgaaggaattttcagaattggagcaaagtggttattatgtctg ctaccccagaggaagcaaaccagaagatgcgaacttttatctctacctgaggg caagagtgtgtgagaactgcatggagatggatgtgatgtcggtggccacaatt gtcatagtggacatctgcatcactgggggcttgctgctgctggtttact actgga gcaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgct ggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaa cccagactatgagcccatccggaaaggccagcgggacctgtattctggcctg aatcagagacgcatctgataagaattcgatccgcggccgcgaaggatctgcg atcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccg agaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggc gcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagg gtggggggagaaccgtatataagtgcagtagtcgccgt gaacgttctttttcgca acgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctcct tcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgc gttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggta agtttaaag ctcaggtcgagaccgggcctttgtccggcgctcccttggagccta cctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacg tctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgccta cgctagatgaccgagtacaagcccacggtgcgcct cgccacccgcgacgac gtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgcc acgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctg caagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcg cggacgacggcgccgcggt a ccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcg gccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaa cctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgc ccgaaggaccgcgcacctggtgcatgacccgcaag cccggtgcctgagtcg

- 68 043737

acaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatg ttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgct tcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag gagttgtggcccgt tgtcaggcaacgtggcgtggtgtgcactgtgtttgctgac gcaacccccactggttggggcattgccaccacctgtcagctcctttccgggact ttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgccc gctgctggacaggggctcggctgttgggcactgacaattccgtgg tgttgtcg gggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcg cgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcc cgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcag acgag tcggatctccctttgggccgcctccccgcctggtacctttaagaccaat gacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggact ggaagggctaattcactcccaacgaaaataagatctgctttttgcttgtactggg tctctctggttagaccagatctgagcctgggagctctctggctaactagggaac cc actgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgccc gtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtgg aaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaa agaaatgaatatcagagagtgagaggaacttgttt attgcagcttataatggtta caaataaagcaatagcatcacaaatttcacaaataaagcattttttcactgcattc tagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcc cgcccctaactccgcccagttccgcccattctccgccccatggctgactaatttt ttttatttatgcagaggccga ggccgcctcggcctctgagctattccagaagta gtgaggaggcttttttggaggcctagacttttgcagagacggcccaaattcgta atcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaa catacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagc taactcacat taattgcgttgcgctcactgcccgctttccagtcgggaaacctgt cgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcg tattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcgg ctgcggcgagcggtatcagctcactcaaagg cggtaatacggttatccacaga atcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaagg ccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccc cctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccg acaggactataaagataccaggcgt ttccccctggaagctccctcgtgcgctct cctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaa gcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccg ctgcg ccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggt

- 69 043737

gctacagagttcttgaagtggtggcctaactacggctacactagaaggacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagc tcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc agcagattacgcgcagaaaaaaaggatct caagaagatcctttgatcttttctac ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaat caatctaaagtatatgagtaaacttggtctgacagttaccaatgcttaatcagt gaggcacc tatctcagcgatctgtctatttcgttcatccatagttgcctgactccc cgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctg caatgataccgcgagacccacgctcaccggctccagatttatcagcaataaac cagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct c catccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaa tagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcg tttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat cccccatgttgtgcaaaaaagcgg ttagctccttcggtcctccgatcgttgtcag aagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattct cttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaa gtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcg tcaat acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaa aacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttc gatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg tttctgggtgagcaaaaacaggaaggcaaa atgccgcaaaaaagggaataa gggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagca tttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataa acaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaag aaaccattattatcatga cattaacctataaaaataggcgtatcacgaggcccttt cgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctccc ggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccg tcaggcgcgtcagcgggtgttggcgggtgtcggggctggctta actatgcg gcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgca cagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctg cgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct ggcgaaagggggatgtgctgcaaggcgattaag ttgggtaacgccagggttt tcccagtcacgacgttgtaaaacgacggccagtgccaagctg 17 amino acid p510_anti- MSLN_SSl_CD3 _e MLLLVTSLLLCELPHPAFLLIPDIQQVQLQQSGPEL EKPGASVKISCKASGYSFTGYTMNWVKQSHGKSL EWIGLITPYNGAS SYNQKFRGKATLTVDKS SSTAY MDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGT

- 70 043737

TVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSAS PGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWI YDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDD ATYYCQQWSKHPLTFGAGTKLEIKAAAGGGGSG GGGSGGGGSLEDGNEEMGGITQTPYKVSISGTTVI LTCPQYPGSEILWQHNDKNIGGDEDDKNIG SDED HLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYL RARVCENCMEMDVMSVATIVIVDICITGGLLLLV YYWSKNRKAKAKPVTRGAGAGGRQRGQNKERP PPVPNPDYEPIRKGQRDLYSGLNQRRI* 18 anti-MSLN light chain amino acid (MHC1445LC.1) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGN TYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSG SGSGTDFTLKITRVEAEDLGVFFCSQSTHVPFTFGS GTKLEIK 19 Light chain DNA vs. MSLN (MHC 1445LC.1) gatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaag cctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacaccta tttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacaa agtttccaaccgattttctggggtcccagacaggttcag tggcagtggatcagg gactgatttcacactcaagatcaccagagtggaggctgaggatctgggagtttt tttctgctctcaaagtacacatgttccattcacgttcggctcggggacaaagttg gaaataaaa 20 heavy chain amino acid against MSLN (MHC1445HC.1) QVQLQQSGAELVRPGASVTLSCKASGYTFFDYEM HWVKQTPVHGLEWIGAIDPEIDGTAYNQKFKGK AILTADKSSSTAYMELRSLTSEDSAVYYCTDYYG SSYWYFDVWGTGTTVTVSS 21 Heavy chain DNA vs. MSLN (MHC 1445NS.1) caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagt gacgctgtcctgcaaggcttcgggctacacattttttgactatgaaatgcactgg gtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctga aattgatggtactgcctacaatcagaagttcaagggcaaggccat actgactgc agacaaatcctccagcacagcctacatggagctccgcagcctgacatctgag gactctgccgtctattactgtacagattactacggtagtagctactggtacttcga tgtctggggcacagggaccacggtcaccgtctcctc 22 anti-MSLN light chain amino acid (MHC1446LC.1) DVMMTQTPLSLPVSLGDQASISCRSSQSLVHSNG NTYLHWFLQKPGQSPKLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPLTF GAGTKLELK 23 Light chain DNA vs. MSLN (MHC 1446LC.1) gatgttatgatgacccaaactccactctccctgcctgtcagtcttggagatcaag cctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacaccta

- 71 043737

tttacattggttcctgcagaagccaggccagtctccaaagctcctgatctacaaa gtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcaggg acagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttat ttctgctctcaaactacacatgttccgctcacgttcgg tgctgggaccaagctgg agctgaaa 24 heavy chain amino acid against MSLN (MHC1446HC.3) QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEM HWVKQTPVHGLEWIGAIDPEIAGTAYNQKFKGK AILTADKSSSTAYMELRSLTSEDSAVYYCSRYGG NYLYYFDYWGQGTTLTVSS 25 Heavy chain DNA against MSLN (MHC1446HC.Z) caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagt gacgctgtcctgcaaggcttcgggctacacttttactgactatgaaatgcactgg gtgaagcagacacctgtccatggcctggaatggattggagctattgatcctgaa attgctggtactgcctacaatcagaagttcaagggcaaggccat actgactgca gacaaatcctccagcacagcctacatggagctccgcagcctgacatctgagg actctgccgtctattactgttcaagatacggtggtaactacctttactactttgact actggggccaaggcaccactctcacagtctcctca 26 anti-MSLN light chain amino acid (MHC1447LC.5) DVLMTQIPLSLPVSLGDQASISCRSSQNIVYSNGNT YLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGS GTKLEIK 27 Light chain DNA against MSLN (MHC1447LC.5) gatgttttgatgacccaaattccactctccctgcctgtcagtcttggagatcaagc ctccatctcttgcagatctagtcagaacattgtgtatagtaatggaaacacctattt agagtggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaag tttccaaccgattttctggggtcccagacaggttcag tggcagtggatcaggga cagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatt actgctttcaaggttcacatgttccattcacgttcggctcggggacaaagttgga aataaaa 28 anti-MSLN heavy chain amino acid (MHC1447HC.5) QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEM HWVKQTPVHGLEWIGAIDPEIGGSAYNQKFKGRA ILTADKSSSTAYMELRSLTSEDSAVYYCTGYDGY FWFAYWGQGTLVTVS S 29 Heavy chain DNA against MSLN (MHC1447HC.5) caggttcaactgcagcagtccggggctgagctggtgaggcctggggcttcag tgacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactg ggtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctg aaattggtggttctgcctacaatcagaagttcaagggcaggggcca tattgactg cagacaaatcctccagcacagcctacatggagctccgcagcctgacatctga ggactctgccgtctattattgtacgggctatgatggttacttttggtttgcttactgg ggccaagggactctctggtcactgtctcttca

- 72 043737

thirty anti-MSLN light chain amino acid (MHC1448LC.4) ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHW YQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNS YSLTISSMEAEDVATYYCFQGSGYPLTFGSGTKLE IK 31 Light chain DNA against MSLN (MHC1448LC.4) gaaaatgttctcacccagtctccagcaatcatgtccgcatctccaggggaaaa ggtcaccatgacctgcagtgctagctcaagtgtaagttacatgcactggtacca gcagaagtcaagcacctcccccaaactctggatttatgacacatccaaactgg cttctggagtcccaggtcgcttcagtggcagtgggtctggaaact cttactctct cacgatcagcagcatggaggctgaagatgttgccacttattactgttttcaggg gagtgggtacccactcacgttcggctcggggacaaagttggaaataaaa 32 anti-MSLN heavy chain amino acid (MHC1448HC.3) QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEM HWVKQTPVHGLEWIGGIDPETGGTAYNQKFKGK AILTADKSSSTAYMELRSLTSEDSAVYYCTSYYGS RVFWGTGTTVTVS S 33 Heavy chain DNA against MSLN (MHC1448HC.3) caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagt gacgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactg ggtgaaacagacacctgtgcatggcctggaatggattggaggtattgatcctg aaactggtggtactgcctacaatcagaagttcaagggtaaggcca tactgact gcagacaaatcctccagcacagcctacatggagctccgcagcctgacatctg aggactctgccgtctattactgtacaagttactatggtagtagagtcttctggggc acagggaccacggtcaccgtctcctca 34 anti-MSLN light chain amino acid (MHC1449LC.3) QIVLSQSPAILSAFPGEKVTMTCRASSSVSYMHWY QQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSY SLTIS SVEAEDAATYYCQQWS SNPPTLTFGAGTKL ELK 35 Light chain DNA against MSLN (MHC1449LC.3) caaattgttctctcccagtctccagcaatcctgtctgcatttccaggggagaagg tcactatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagc agaagccaggatcctcccccaaaccctggatttatgccacatccaacctggctt ctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactct ctcac aatcagcagtgtggaggctgaagatgctgccacttattactgccagcagtgga gtagtaacccaccacgctcacgttcggtgctgggaccaagctggagctgaa a 36 heavy chain amino acid against MSLN (MHC1449HC.3) QVQLQQSGAELARPGASVKLSCKASGYTFTSYGI SWVKQRTGQGLEWIGEIYPRSGNTYYNESFKGKV TLTADKSSGTAYMELRSLTSEDSAVYFCARWGSY GSPPFYYGMDYWGQGTSVTVSS 37 Heavy chain DNA against MSLN (MHC1449HC.Z) caggttcagctgcagcagtctggagctgagctggcgaggcctggggcttcag tgaagctgtcctgcaaggcttctggctacaccttcacaagctatggtataagctg

- 73 043737

t tactatggtatggactactggggtcaaggaacctcagtcaccgtctcctca 38 anti-MSLN light chain amino acid (MHC1450LC.3) DVLMTQTPLSLPVSLGNQASISCRSSQSIVHSSGST YLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFG GGTKLEIK 39 Light chain DNA against MSLN (MHC1450LC.3) gatgttttgatgacccaaactccactctccctgcctgtcagtcttggaaatcaag cctccatctcttgcagatctagtcagagcattgtacatagtagtggaagcaccta tttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaa agtttccaaccgattttctggggtcccagacaggttcag tggcagtggatcagg gacagatttcacactcaagatcagcagagtggaggctgaggatctgggagttt attactgctttcaaggctcacatgttccatacacgttcggaggggggaccaagc tggaaataaaa 40 anti-MSLN heavy chain amino acid (MHC1450HC.5) QVQLQQSGAELARPGTSVKVSCKASGYTFTSYGI SWVKQRIGQGLEWIGEIHPRSGNSYYNEKIRGKA TLTADKSSSTAYMELRSLISEDSAVYFCARLITTV VANYYAMDYWGQGTSVTVSS 41 Heavy chain DNA against MSLN (MHC1450HC.5) caggttcagctgcagcagtctggagctgagctggcgaggcctgggacttcag tgaaggtgtcctgcaaggcttctggctataccttcacaagttatggtataagctg ggtgaagcagagaattggacagggccttgagtggattggagagattcatccta gaagtggtaatagttactataatgagaagatcaggggcaaggccacactgact gcagacaaatcctccagcacagcgtacatggagctccgcagcctgatatctga ggactctgcggtctatttctgtgcaaggctgattactacggtagttgctaattact atgctatggactactggggtcaaggaacctcagtcaccgtctcctca 42 anti-MSLN light chain amino acid (MHC1451LC.1) DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTR KNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFT GSGSGTDFTLTISSVQAEDLAVYYCKQSYNLVTF GAGTKLELK 43 Light chain DNA against MSLN (MHC 1451 LC.1) gacattgtgatgtcacagtctccatcctccctggctgtgtcagcaggagagaag gtcactatgagctgcaaatccagtcagagtctgctcaacagtagaacccgaaa gaactacttggcttggtaccagcagaaaccagggcagtctcctaaactgctgat ctactgggcatccactagggaatctggggtccctgatcgcttcacagg cagtg gatctgggacagatttcactctcaccatcagcagtgtgcaggctgaagacctg

- 74 043737

gcagtttattactgcaaacaatcttataatctggtcacgttcggtgctgggaccaa gctggagctgaaa 44 heavy chain amino acid against MSLN (MHC 1451HC.2) QVQLQQSGAELVRPGASVTLSCKASGYTFFDYEM HWVKQTPVHGLEWIGAIDPEIDGTAYNQKFKGK AILTADKSSSTAYMELRSLTSEDSAVYYCTDYYG SSYWYFDVWGTGTTVTVSS 45 Heavy chain DNA vs. MSLN (MHC 1451NS.2) caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagt gacgctgtcctgcaaggcttcgggctacacattttttgactatgaaatgcactgg gtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctga aattgatggtactgcctacaatcagaagttcaagggcaaggccat actgactgc agacaaatcctccagcacagcctacatggagctccgcagcctgacatctgag gactctgccgtctattactgtacagattactacggtagtagctactggtacttcga tgtctggggcacagggaccacggtcaccgtctcctc 46 anti-MSLN light chain amino acid (MHC1452LC.1) QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWY QQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSY SLTISSMEAEDAAIYYCQQYHSYPLTFGAGTKLE LK 47 Light chain DNA against MSLN (MHC 1452LC.1) caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaag gtcaccatatcctgcagtgccagctcaagtgtaagttacatgtactggtaccag cagaagccaggatcctcccccaaaccctggatttatcgcacatccaacctggc ttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctct ca caatcagcagcatggaggctgaagatgctgccacttattactgccagcagtatc atagttacccactcacgttcggtgctgggaccaagctggagctgaaa 48 anti-MSLN light chain amino acid (MHC1452LC.6) QIVLTQSPAIMSASPGERVTMTCSASSSVSSSYLY WYQQKSGSSPKLWIYSISNLASGVPARFSGSGSGT SYSLTINSMEAEDAAIYYCQQWSSNPQLTFGAGT KLELK 49 Light chain DNA against MSLN (MHC1452LC.6) caaattgttctcacccagtctccagcaatcatgtctgcatctcctggggaacgg gtcaccatgacctgcagtgccagctcaagtgtaagttccagctacttgtactggt accagcagaagtcaggatcctccccaaaactctggatttatagcatatccaacc tggcttctggagtcccagctcgcttcagtggcagtgggtctgggacct cttactc tctcacaatcaacagcatggaggctgaagatgctgccacttattactgccagca gtggagtagtaacccacagctcacgttcggtgctgggaccaagctggagctg aaa 56 heavy chain amino acid against MSLN (MHC1452HC.2) QVQLKQSGAELVKPGASVKISCKASGYTFTDYYI NWVKQRPGQGLEWIGKIGPGSGSIYYNEKFKGK ATLTADKSSSTAYMQLSSLTSEDSAVYFCARTGY YVGYYAMDYWGQGTSVTVSS

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Claims (3)

50 Heavy chain DNA against MSLN (MHC1452HC.2) caggtccagctgaagcagtctggagctgagctggtgaagcctggggcttcag tgaagatatcctgcaaggcttctggctacaccttcactgactactatataaactg ggtgaagcagaggcctggacagggccttgagtggattggaaagattggtcct ggaagtggtagtacttact acaatgagaagttcaagggcaaggccacactgac tgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctg aggactctgcagtctatttctgtgcaagaactggttactacgttggttactatgcta tggactactggggtcaaggaacctcagtcaccgtctcctca 51 amino acid heavy chain anti-MSLN (MHC14 52HC.4) QVQLQQSGAELARPGASVKLSCKASGYTFTIYGIS WVKQRTGQGLEWIGEIYPRSDNTYYNEKFKGKA TLTADKSSSTAYMELRSLTSEDSAVYFCARWYSF YAMDYWGQGTSVTVSS 52 Heavy chain DNA against MSLN (MHC1452HC.4) caggttcagctgcagcagtctggag ctgagctggcgaggcctggggcttcag tgaagctgtcctgcaaggcttctggctacaccttcacaatctatggtataagctg ggtgaaacagagaactggacagggccttgagtggattggagagatttatccta gaagtgataatacttactacaatgagaagttcaagggcaaggccacactgact gcagacaaatcctccagcacagcgtacat ggagctccgcagcctgacatctg aggactctgcggtctatttctgtgcaagatggtactcgttctatgctatggactac tggggtcaaggaacctcagtcaccgtctcctca 58 Single domain binding antibody 1 against MSLN (SD1) EVQLVESGGGLVQPGGSLRLSCAASGGDWSANF MYWYRQAPGKQRELVARISGRGVVDYVESVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAVASY WGQGTLVTVSS 59 Single domain binding anti-MSLN binding antibody 4 (SD4) EVQLVESGGGLVQPGGSLRLSCAASGSTSSINTMY WYRQAPGKERELVAFISSGGSTNVRDSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCNTYIPYGG TLHDFWGQGTLVTVSS 55 Anti-MSLN single-domain binding antibody 6 (SD6) QVQLVESGGGVVQAGGSLRLS CAASGSTFSIRAM RWYRQ APGTERDLVA VIYGS STYYAD A VKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGT ARDYWGQGTLVTVSS FORMULA OF THE INVENTION 1. A recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) containing: (a) a TCR subunit comprising (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from a CD3 epsilon, CD3 delta, or CD3 gamma intracellular signaling domain; and (b) a murine, human, or humanized antibody variable domain comprising an anti-mesothelin binding domain, wherein the murine, human, or humanized antibody variable domain is a scFv or sdAb antibody domain; wherein the TCR extracellular domain and the anti-mesothelin binding domain are operably linked, wherein at least two of (i), (ii) and (iii) are derived from the same TCR subunit, wherein the same TCR subunit is CD3 epsilon, CD3 delta or CD3 gamma, and TFP functionally interacts with the TCR when expressed in the T cell. 2. The nucleic acid molecule according to claim 1, wherein the anti-mesothelin binding domain is operably linked to the TCR extracellular domain via a linker sequence. 3. The nucleic acid molecule according to claim 2, in which the linker sequence contains (G4S)n, where n = from 1 to 4. -