Humanized SCFV antibody targeting the proximal region of CD30 for Anti-cancer cellular therapy

Abstract

Disclosed herein is a fully humanized single chain variable fragment (scFV) which targets the proximal portion of the CD30 molecule, and which can be used as part of an immunotherapeutic strategy for cancer treatment. The disclosed scFV avoids binding by soluble CD30 and is significantly less immunogenic and thus more likely to persist and provide durable responses in the context of cancer immunotherapy.

Description

HUMANIZED SCFV ANTIBODY TARGETING THE PROXIMAL REGION OF CD30 FOR ANTI-CANCER CELLULAR THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 605,743, filed December 4, 2023, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “321502-2020 Sequence Listing” created on December 2, 2024, and having 23,544 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] CD30 is a transmembrane protein that has been used as an immunotherapeutic target to treat a variety of cancer types (e.g. Hodgkin’s Lymphoma, NonHodgkin’s Lymphoma, and T-cell and NK cell malignancies). Classic Hodgkin’s Lymphoma (cHL) in particular is a disease subtype that is in great need of novel therapies; approximately 9200 new adult cases (and 1100 new pediatric cases) are diagnosed each year (approximately 11% of all lymphoma cases in the U.S.) with 900 deaths attributed to cHL annually. Though many patients with cHL can achieve durable remission with first-line therapy, up to 15-40% will ultimately relapse or progress. Next line standard of care is high- dose chemotherapy followed by autologous stem cell transplantation (aSCT), however half of patients relapse after transplantation and many patients are not candidates for auto transplant.

[0004] Hodgkin / Reed-Sternberg (HRS) cells universally express CD30. CD30 is a transmembrane protein that has proven to be an effective and safe immunotherapeutic target to treat a variety of cancer types (ex. Hodgkin’s Lymphoma, Non-Hodgkin’s Lymphoma, T-cell and NK cell malignancies). The CD30-specific antibody drug conjugate brentuximab vedotin (BV) is active in HL but offers sustained remissions in fewer than a quarter of patients with relapsed or refractory (r / r) disease. Advances have also been made with checkpoint inhibitors as well, however the majority of patients who progress after aSCT, including those who receive BV and anti-PD-1 therapies, will still ultimately fail to achieve durable remission. The prognosis for these individuals is dismal, with allogeneic stem cell transplantation (alloSCT) traditionally remaining the only curative option but comes at the cost of high morbidity and mortality associated with it.

[0005] A chimeric antigen receptor (CAR) T-cell (CART) product containing a murine scFV which recognizes the distal portion of the extracellular domain of CD30 has been used to treat cHL. Clinical responses were obtained with this distal targeting CD30 CAR-T product, however persistence of the CAR-T was extremely poor compared to CD19 targetingCAR-T products which contain similar CAR-T construct elements. It is known that the distal portion (i.e. the extra-cellular domain) of the CD30 molecule can be shed from cells and circulate as soluble CD30. It has been hypothesized that for this CAR-T targeting the distal portion of CD30, circulating soluble CD30 present in patients’ blood bound to infused CD30 targeting CAR T-cells and resulted in their subsequent elimination from circulation.

[0006] Of note, the scFV comprising the antigen binding portion of CAR molecules and targeting antigens of interest are often composed of murine elements. Host immune anti-murine CAR responses can also result in host rejection of CAR T-cells and reduction in long-term persistence of the CAR (PMID: 29678657). In the case of the clinical trial utilizing the distal targeting CD30 CART, the scFV targeting CD30 was also murine. Thus, it was felt that both binding of the distal CD30 targeting scFV of the CAR by soluble CD30 as well as immune rejection of the murine CD30 scFV resulted in further enhanced removal of the CD30 CAR-T from circulation and a lack of CART persistence. Ultimately, the poor persistence resulted in relapse for the vast majority of patients treated with the distal targeting CD30 CART, including those who initially had achieved complete responses. It is important to note, that that murine scFV’s are currently used in CD19 targeting commercial CART agents, with good success (ex. Axicabtagene ciloleucel, Tisagenlecleucel). These scFV’s are functional, bind well and provide good responses and in some cases can persist. However, some percent of patients will still reject and have poor persistence. Immunogenicity is patient dependent, presented by certain HI_A subtypes, and therefore this creates a problem of unpredictability in terms of response - some patients may develop antiCAR immune rejection, and some may not. Humanization of scFV’s can significantly reduce immunogenicity and clearance..SUMMARY OF THE INVENTION

[0007] Disclosed herein is a fully humanized single chain variable fragment (scFV) which targets the proximal portion of the CD30 molecule, and which can be used as part of an immunotherapeutic strategy for cancer treatment. The disclosed scFV avoids binding by soluble CD30 and is significantly less immunogenic and thus more likely to persist and provide durable responses in the context of cancer immunotherapy.

[0008] Published literature was first to identify an epitope of the CD30 protein (Figure 1) contained within the membrane bound portion of CD30 proximal to the cleavage site, and not shed as soluble CD30 (Nagata et al; PMID: 15905329). This epitope is much more efficiently presented on membrane bound, cell associated, CD30 than on soluble CD30 and is considered a membrane specific epitope. This is an ideal target for immunotherapy as it allows for CD30 targeting and avoids unwanted binding to soluble CD30. A panel of antibodies / immunotoxin was used to demonstrate how soluble CD30 can limit binding to epitope I, III and IV of CD30. They found that antibodies that target epitopes Ila, V and VI ofCD30 are less affected by soluble CD30. Among those antibodies targeting II, and VI, T105 was found to be preferred in the context of immunotoxin therapy and had improved binding to CD30 even in the presence of soluble CD30 (Patent # US7470775B2).

[0009] The sequence for the T105 mAb clone was obtained from W02007040653A2 and US7470775B2. In order to further improve on the T 105 clone in terms of immunogenicity and functionality (which are required to translate into CAR-T therapy), humanization and optimization analyses was performed utilizing the amino acid sequence for the murine T105 mab (Figure 2). In the humanness scores analysis, the murine T105 heavy chain and light chain regions 1-7 were all significantly below the threshold to meet the criteria for humanized with a score of 0.000 vs 0.5 (Table 1 and Table 2). Utilizing an in-silico approach (Figure 2) and with repeated iterations of mutations of a variety of elements of the T 105 clone, the aim was to develop a novel, humanized design for a proximally targeting CD30 targeting scFV to provide both the least immunogenic and highest functioning.

[0010] In particular, a focus was placed on mutations to remove murine elements from the framework (or backbone) regions of both heavy and light chains in order to reduce immunogenicity (Figure 2). Critical elements within the binding pocket of the protein were preserved in order to preserve functionality. After in silico humanization and with keeping parental complementarity determining regions (CDRs) intact, there was improvement in the humanness score of the novel antibody construct. Humanness is defined as the similarity between the sequences contained within an antibody and known human antibody sequences.

[0011] Increased humanness has been shown to directly correlates with decreased immunogenicity and reduced formation of anti-product antibody formation and immune rejection by the patient. Specifically, an OASis identity score (humanness score) of the original T105 of the 9-mer peptide considered human was 38 % (OASis identity) and was subsequently increased to 77% (Figure 3). An anti-CD30 scFV-CD30 antigen docking model was also created and refined with alanine scanning. For immunogenicity optimization, MHC class I immunogenicity engineering was utilized. Humanized amino acid sequences was used for immunogenicity testing and identified no peptides that were immunogenic on the heavy chains. The I54P amino acid residue was mutated in the framework region 2 to reduce immunogenicity. Humanization of clone T25 (which targets the distal portion of CD30) was also repeated to be used as a control for downstream assays.

[0012] Ultimately, a fully humanized clone, henceforth called OSU-185, was developed that targets the proximal, membrane bound portion of CD30 and contains optimal characteristics in terms of both reduced immunogenicity and retained functionality. Of critical importance, the OSU-185 clone has 91% homology to the T105 clone (Figure 4). However, in-silico analysis revealed significant functional differences including significantly reducedimmunogenicity (from -1.00274 to -0.59594 for heavy chain and -2.09543 to -1.80128 for light chain), increased humanness (Figure 3) and preserved functionality.

[0013] In some embodiments, the OSU-185 scFv comprises a variable heavy (VH) domain having the amino acid sequence QVTLVQSGPEVKQPSATLSVTCQFSGFSLSTSGMGVSWIRQPSGQDLEWLAHIYWDDDKR YAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSS (SEQ ID NO:1), QVTLVQSGPEWQPSATLSLTCQFSGFSLSTSGMGVSWIRQPSGQDLEWLAHIYWDDDKR YAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSS (SEQ ID NO:15), QVTLKASGAGVKQPGATVSVTCKFSGFSLSTSGMGVSWIRQPSGKDLEWLAWIYWDDDK RYAPSLQSRVTITKDTSSNQVFMKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVS S (SEQ ID NO:16), or QVTLKASGAGVLQPGATLSLTCKFSGFSLSTSGMGVSWIRQPSGKDLEWLAWIYWDDDKR YAPSLQSRLTITKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSS (SEQ ID NO:17), having CDR1 , CDR2 and CDR3 sequences, wherein the CDR1 sequence of theHdomain comprises the amino acid sequence GFSLSTSGMGVSWI (SEQ ID NO:3); the CDR2 sequence of the VHdomain comprises the amino acid sequence HIYWDDDKRY (SEQ ID NO:4); the CDR3 sequence of the VHdomain comprises the amino acid sequence ARRADGLYFYLDVW (SEQ ID NO:5). In some embodiments, the OSU-185 scFv VHdomain is a variant having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1 , 15, 16, or 17, while maintaining the CDR1 sequence SEQ ID NO:3, the CDR2 sequence SEQ ID NO:4, and the CDR3 sequence SEQ ID NO:4. In some embodiments, the variant V domain also maintains at least 5V, 6Q, 10E, 11 V, 12K, 16A, 20V, 23Q, 45Q, 62A, and 66L.

[0014] In some embodiments, the OSU-185 scFv comprises a variable light (VL) domain having the amino acid sequence DIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQKPGQAPEALPYSASYLYSGVP SRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQGTKLEIK (SEQ ID NO:2), DIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQKPGQAPEALIYSASYCYSGVP SRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQGTKLEIK (SEQ ID NO: 18), DIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVNWYQQKPGQAPEALIYSASYLYSGVPS RFTGSGSGTDFTLTISNVQSEDLATYFCQQYNSYPLTFGQGTKLEIK (SEQ ID NO:19), or DIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVNWYQQKPGQAPEALIYSASYCYSGVP SRFTGSGSGTDFTLTISNVQSEDLATYFCQQYNSYPLTFGQGTKLEIK (SEQ ID NQ:20), having CDR1 , CDR2 and CDR3 sequences, wherein the CDR1 sequence of the VLdomaincomprises the amino acid sequence QNVNTNVAAWY (SEQ ID NO:6); the CDR2 sequence of the VLdomain comprises the amino acid sequence YSASY (SEQ ID NO:7); the CDR3 sequence of the VLdomain comprises the amino acid sequence QQYNSYPLTF (SEQ ID NO:8). In some embodiments, the OSU-185 scFv VL domain is a variant having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2, 18, 29, or 20, while maintaining the CDR1 sequence SEQ ID NO:6, the CDR2 sequence SEQ ID NO:7, and the CDR3 sequence SEQ ID NO:8. In some embodiments, the variant VLdomain also maintains at least Q3, 9S, 24R, 42A, 48P, 54L, 60S, 80P, 85T, and 100Q.

[0015] The heavy and light chains are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:9).

[0016] Therefore, in some embodiments, the scFv comprises the amino acid sequence DIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQKPGQAPEALPYSASYLYSGVP SRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQGTKLEIKGGGGSGGGGSGG GGSQVTLVQSGPEVKQPSATLSVTCQFSGFSLSTSGMGVSWIRQPSGQDLEWLAHIYWD DDKRYAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVT VSS (SEQ ID NO: 10), QVTLVQSGPEVKQPSATLSVTCQFSGFSLSTSGMGVSWIRQPSGQDLEWLAHIYWDDDKR YAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSSG GGGSGGGGSGGGGSDIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQKPGQA PEALPYSASYLYSGVPSRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQGTKL EIK (SEQ ID NO:11), QVTLVQSGPEWQPSATLSLTCQFSGFSLSTSGMGVSWIRQPSGQDLEWLAHIYWDDDKR YAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSSG GGGSGGGGSGGGGSDIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQKPGQA PEALIYSASYCYSGVPSRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQGTKLE IK (SEQ ID NO:21), QVTLKASGAGVKQPGATVSVTCKFSGFSLSTSGMGVSWIRQPSGKDLEWLAWIYWDDDK RYAPSLQSRVTITKDTSSNQVFMKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVS SGGGGSGGGGSGGGGSDIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVNWYQQKPG QAPEALIYSASYLYSGVPSRFTGSGSGTDFTLTISNVQSEDLATYFCQQYNSYPLTFGQGTK LEIK (SEQ ID NO:22), or QVTLKASGAGVLQPGATLSLTCKFSGFSLSTSGMGVSWIRQPSGKDLEWLAWIYWDDDKR YAPSLQSRLTITKDTSSNQVFLKITSVDTADTATYYCARRADGLYFYLDVWGAGTTVTVSSG GGGSGGGGSGGGGSDIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVNWYQQKPGQAPEALIYSASYCYSGVPSRFTGSGSGTDFTLTISNVQSEDLATYFCQQYNSYPLTFGQGTKLE IK (SEQ ID NO:23), or or variants thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:10, 11 , 21 , 22, or 23, while maintaining the VH CDR1 sequence SEQ ID NO:3, the VHCDR2 sequence SEQ ID NO:4, and the VHCDR3 sequence SEQ ID NO:5 and the VL CDR1 sequence SEQ ID NO:6, the VL CDR2 sequence SEQ ID NO:7, and the VL CDR3 sequence SEQ ID NO:8

[0017] Subsequently, a second-generation chimeric antigen receptor (CAR) construct was designed with 4-1 BB and CD3^ chain signaling domains by joining the novel humanized scFV specifically targeting the proximal, membrane bound epitope of CD30 (ep2) with a CD8 hinge / transmembrane domain and CD3 (Figure 5). The sequence was then codon-optimized, synthesized and subcloned into a lentiviral vector pCDH. This vector was used to generate humanized, proximal CD30 targeting CAR T-cells (OSU-185-CAR30) from healthy donor mononuclear cell products, according to the usual methods.

[0018] Therefore, also disclosed herein is a CAR polypeptide having a CD30 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory signaling region, wherein the CD30 antigen binding domain comprises the fully humanized scFv disclosed herein.

[0019] In some embodiments, the costimulatory signaling region comprises the cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1 BB, 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof. In some embodiments, the intracellular signaling domain comprises a CD3 zeta (CD3Q signaling domain.

[0020] For example, in some embodiments, the QSU-185-CAR30 polypeptide has the amino acid sequence MALPVTALLLPLALLLHAARPDIQMTQSQSFMSTSVGDRVSVTCRASQNVNTNVAWYQQK PGQAPEALPYSASYLYSGVPSRFTGSGSGTDFTLTISNVQPEDLATYFCQQYNSYPLTFGQ GTKLEIKGGGGSGGGGSGGGGSQVTLVQSGPEVKQPSATLSVTCQFSGFSLSTSGMGVS WIRQPSGQDLEWLAHIYWDDDKRYAPSLLSRLTISKDTSSNQVFLKITSVDTADTATYYCAR RADGLYFYLDVWGAGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO:12).

[0021] QSU-185-CAR30 cells were then used in-vitro for cytotoxicity analysis to assess for specificity and functionality (Figure 6). OSU-185-CAR30 cells containing theOSU-185 scFV, CAR T-cells containing an scFV comprised of the T25 clone which targets the distal, extracellular domain of CD30, and non-transduced T-cell controls (NTT’s) were tested. Cell lines lacking expression of CD30 were used as controls and those strongly expressive of CD30, including the well-known Hodgkin’s lymphoma cell lines L540 and L428 to demonstrate that OSU-185-CAR30 has significantly increased cytotoxicity compared to T25 containing CAR T-cells and untransduced T-cells (UTT’s) for CD30 expressing Hodgkin’s lymphoma cell lines (e.g. L540 and L428). OSU-185-CAR30 cells also had reduced killing of non-specific CD30 lacking cancer cell lines (e.g. Mino and DC9) compared to T25 CAR T-cells, with killing similar to UTT’s. Of note, L540 and L428 are Hodgkin’s Lymphoma cell lines which produce high amounts of soluble CD30. Thus OSU-185-CAR30 cells outperform OSU-184-CAR30 cells in the presence of soluble CD30. The lack of binding to soluble CD30 by OSU-185-CAR30 cells compared to the distal targeting OSU-184- CAR30’s, containing CART constructs which targeted more distal element of CD30 and can be found by soluble CD30, though more studies to confirm this are required.

[0022] There are a multitude of applications for the disclosed humanized scFV which targets the proximal portion of CD30. One embodiment includes the development of a CAR T-cell product used to treat Hodgkin’s Lymphoma in adults and pediatrics. cHL is an entity that currently has poor long term outcomes for the majority of patients that fail induction therapy and relapse after or are ineligible for auto transplant. Allogeneic transplant remains the only curative option for the majority of these patients, however this carries with it significant morbidity, mortality, and long term quality of life implications. Prior large studies in the U.S. utilizing CD30 targeting CAR T-products targeted the distal, extracellular domain of CD30 were marked by lack of persistence and high rates of relapse. To date, there are no CAR-T studies open in the U.S. utilizing this strategy of targeting the proximal, membrane bound for Hodgkin’s lymphoma. There are currently no approved cell therapy products for cHL, however Hodgkin’s lymphoma has proven amenable to achieve responses with CAR-T therapy, though persistence has remained the critical issue to address.

[0023] Therefore, also disclosed herein is a method of providing an anti-cancer immunity in a subject with a CD30-expressing cancer, the method comprising administering to the subject an effective amount of an immune effector cell genetically modified to express the CAR polypeptide of any one of claims 2 to 4, thereby providing an anti-tumor immunity in the mammal. In some embodiments, the CD30-expressing cancer is Hodgkin’s Lymphoma, Non-Hodgkin’s Lymphoma. In some embodiments, the CD30-expressing cancer is Classic Hodgkin’s Lymphoma (cHL).

[0024] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0025] Figure 1 shows topographical epitopes (Ep) of T105 and T25 on CD30 molecules.

[0026] Figure 2 shows humanization of T105 heavy (SEQ ID NO: 13) and light chain (SEQ ID NO:14) elements yields OSU-185 heavy (SEQ ID NO:1) and light (SEQ ID NO:2) chains.

[0027] Figure 3 shows substantial increase in OASis identity and germline content with antibody humanization reveals increase in the humanness score of OSU-185.

[0028] Figures 4Ato 4B show pairwise protein sequence alignment between T105 and final humanized OSU-185 sequence.

[0029] Figures 5A and 5B show design of a CD30 CAR lentiviral vectors.

[0030] Figure 6 shows expression of CD30 on classical Hodgkin’s Lymphoma (cHL) and control cell lines.

[0031] Figure 7 shows efficient transduction with the OSU-184 and OSU-185 CD30 CAR vectors.

[0032] Figure 8 shows cytotoxicity analysis of OSU-184 and OSU-185 CD30 CAR-T cells against 2 classical Hodgkin lymphoma (cHL) cell lines: L-428 and L-540. Ns: not significant; ** p<0.01 ; ****p<0.0001 .

[0033] Figure 9 shows immunogenicity of parental and humanized CD30 CARs. Protein lysates of CD30 CAR-T cells containing the T25 , T105, OSU-184, and OSU-185 single chain variable fragments were prepared, fragmented and pulsed with human peripheral blood mononuclear cells (PBMCs). PBMCs contained antigen presenting cells and T cells at 100 pg / mL. The pulsed PBMCs were then cultured in the presence of low dose IL-2 for 6 days before they were harvested and stained for CD25 (T cell activation) by flow cytometry. Induced CD25 expression (as mean fluorescence intensity) was normalized with isotype staining and un-pulsed T cell controls.

[0034] Figures 10A and 10B show OSU-185 CD30 CAR-T cells significantly reduced the tumor growth as detected by IVIS imaging and tumor size measurements on day 14. 500,000 luciferased L-540 cells were engrafted subcutaneously near the flanks of the animals. At Day 3, tumor engraftment was confirmed by IVIS imaging and then mice were randomized into 4 groups: PBS (negative untreated control, blue, n=4), UTT (untransduced T cells, n=5), T25 CD30 CAR-T cells (n=5), and OSU-185 CD30 CAR-T cells (n=5). Fig. 10A shows bioluminescence and FIG. 10B shows tumor sizes from the tumors at Day 14 in each group of mice were plotted as mean ± SEM.DETAILED DESCRIPTION

[0035] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and assuch may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0038] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0039] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0040] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0041] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0042] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0043] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0044] The term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0045] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0046] The term “antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, Fc, and Fd fragments. The antibody fragment may be produced by any means. Forinstance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced.The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

[0047] The term “antigen binding site” refers to a region of an antibody that specifically binds an epitope on an antigen.

[0048] The term “aptamer” refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity. A “nucleic acid aptamer” is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. A “peptide aptamer” is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.

[0049] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0050] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecules is a fusion protein.

[0051] The term “engineered antibody” refers to a recombinant molecule that comprises at least an antibody fragment comprising an antigen binding site derived from the variable domain of the heavy chain and / or light chain of an antibody and may optionally comprise the entire or part of the variable and / or constant domains of an antibody from any of the Ig classes (for example IgA, IgD, IgE, IgG, IgM and IgY).

[0052] The term “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically. A monoclonal antibody binds preferentially to a single specific epitope of a molecule that can be molecularly defined. In the present invention, multiple epitopes can be recognized by a multispecific antibody.

[0053] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0054] The term “Fab fragment” refers to a fragment of an antibody comprising an antigen-binding site generated by cleavage of the antibody with the enzyme papain, which cuts at the hinge region N-terminally to the inter-H-chain disulfide bond and generates two Fab fragments from one antibody molecule.

[0055] The term “F(ab')2 fragment” refers to a fragment of an antibody containing two antigen-binding sites, generated by cleavage of the antibody molecule with the enzyme pepsin which cuts at the hinge region C-terminally to the inter-H-chain disulfide bond.

[0056] The term “Fc fragment” refers to the fragment of an antibody comprising the constant domain of its heavy chain.

[0057] The term “Fv fragment” refers to the fragment of an antibody comprising the variable domains of its heavy chain and light chain.

[0058] “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which is otherwise transcribable to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc), may be transfected into cells, e.g. in certain embodiments mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct. The gene construct may include one or more regulatory elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0059] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at thatposition. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0060] The term “linker” is art-recognized and refers to a molecule or group of molecules connecting two compounds, such as two polypeptides. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.

[0061] The term “multivalent antibody” refers to an antibody or engineered antibody comprising more than one antigen recognition site. For example, a “bivalent” antibody has two antigen recognition sites, whereas a “tetravalent” antibody has four antigen recognition sites. The terms “monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, a “monospecific” antibody's antigen recognition sites all bind the same epitope. A “bispecific” antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A “multivalent monospecific” antibody has multiple antigen recognition sites that all bind the same epitope. A “multivalent bispecific” antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

[0062] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0063] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0064] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0065] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0066] The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that of the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and / or an interaction site of the reference polypeptide. In another embodiment, a fragment may have immunogenic properties.

[0067] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0068] The term “single chain variable fragment or scFv” refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked. One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of aheavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.

[0069] A “spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0070] The term “specifically binds”, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M-1(e.g., 106M-1, 107M-1, 108M~1, 109M~1, 1010M~1, 1011M~1, and 1012M~1or more) with that second molecule.

[0071] The term “specifically deliver” as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule.

[0072] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0073] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0074] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0075] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0076] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0077] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).Antibodies

[0078] Antibodies that can be used in the disclosed compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largelyadopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0079] Also disclosed are fragments of antibodies which have bioactivity. The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.

[0080] Techniques can also be adapted for the production of single-chain antibodies specific to an antigenic protein of the present disclosure. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody can be created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation.

[0081] Divalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VHand two VL regions, yielding tandem scFvs. ScFvs can also be designed with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers (triabodies or tribodies). Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.

[0082] A bi-specific antibody designed to selectively bind CD3 and CD30 would trigger non-specific T-cell activation & cytokine storm. A bi-specific diabody designed to selectively bind CD3 and CD30 would have a molecular weight (55-60 kD) less than the renal clearance threshold, which would result in rapid elimination. As such, diabodies must be administered by a continuous infusion. The disclosed tetravalent, bi-specific antibody can have a molecular weight (e.g., 105-110 kD) greater than the renal filtration threshold with markedly extended PK.

[0083] Provided are fusion polypeptides capable of forming a multivalent engineered antibody that is able to engage T-cells to destroy CD30-expressing malignant cells. Theengineered antibody may comprise for example, at least one scFv, at least one Fab fragment, at least one Fv fragment, etc. It may be bivalent, trivalent, tetravalent, etc. The multivalent antibodies is multispecific, e.g., bispecific, trispecific, tetraspecific, etc. The multivalent antibodies may be in any form, such as a diabody, triabody, tetrabody, etc.

[0084] Bivalent and bispecific antibodies can be constructed using only antibody variable domains. A fairly efficient and relatively simple method is to make the linker sequence between the VH and VL domains so short that they cannot fold over and bind one another. Reduction of the linker length to 3-12 residues prevents the monomeric configuration of the scFv molecule and favors intermolecular VH-VL pairings with formation of a 60 kDa non-covalent scFv dimer “diabody”. The diabody format can also be used for generation of recombinant bis-pecific antibodies, which are obtained by the noncovalent association of two single-chain fusion products, consisting of the VH domain from one antibody connected by a short linker to the VL domain of another antibody. Reducing the linker length still further below three residues can result in the formation of trimers (“triabody”, about 90 kDa) or tetramers (“tetrabody”, about 120 kDa). For a review of engineered antibodies, particularly single domain fragments, see Holliger and Hudson, 2005, Nature Biotechnology, 23:1126-1136. All of such engineered antibodies may be used in the fusion polypeptides provided herein. Tetravalent Tandab® may be prepared substantially as described in WO 1999057150 A3 or US20060233787, which are incorporated by reference for the teaching of methods of making Tandab® molecules.

[0085] The antigen recognition sites or entire variable regions of the engineered antibodies may be derived from one or more parental antibodies directed against any antigen of interest (e.g., CD30). The parental antibodies can include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using sequences of antibodies or antibody fragments known to be specific for an antigen of interest. Sequences that may be derived from parental antibodies include heavy and / or light chain variable regions and / or CDRs, framework regions or other portions thereof.

[0086] Multivalent, multispecific antibodies may contain a heavy chain comprising two or more variable regions and / or a light chain comprising one or more variable regions wherein at least two of the variable regions recognize different epitopes on the same antigen.

[0087] Candidate engineered antibodies for inclusion in the fusion polypeptides, or the fusion polypeptides themselves, may be screened for activity using a variety of known assays. For example, screening assays to determine binding specificity are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow etal. (Eds.), ANTIBODIES: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y., 1988, Chapter s.Pharmaceutical composition

[0088] Also disclosed is a pharmaceutical composition comprising a disclosed molecule in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0089] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0090] Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0091] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.Methods of Treatment

[0092] Also disclosed is a method for treating a CD30-expressing cancer in a subject by administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition. The method can further involve administering to the subject a chemotherapy such as fludarabine, cytarabine, cyclophosphamide, idarubicin, daunorubicin, or a targeted inhibitor such as imbruvica, midostaurin, idelalisib, or an immune agent such as PD1 or PDLl inhibitors.

[0093] The disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.

[0094] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0095] The compositions disclosed herein may be administered prophylactically to patients or subjects who are at risk for a CD30-expressing cancer. Thus, the method can further comprise identifying a subject at risk for a CD30-expressing cancer prior to administration of the herein disclosed compositions.

[0096] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desiredeffect in which the symptoms disorder is affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical daily dosage of the disclosed composition used alone might range from about 1 pg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

[0097] In some embodiments, the molecule is administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1 pig to about 100 mg per kg of body weight, from about 1 pg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of molecule containing lenalidomide administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 pg, 10 pg, 100 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.CD30-specific chimeric antigen receptors (CAR)

[0098] CARs generally incorporate an antigen recognition domain from the singlechain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45). Disclosed herein is a CD30-specific chimeric antigen receptor (CAR) that can be that can be expressed in immune effector cells to enhance antitumor activity against CD30- specific CARs.

[0099] The disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain comprises the CD30-binding region and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain (TD), is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. For example, the endodomain can contain a signaling domain (ISD) and a co-stimulatory signaling region (CSR).

[0100] A “signaling domain (SD)” generally contains immunoreceptor tyrosine-based activation motifs (ITAMs) that activate a signaling cascade when the ITAM is phosphorylated. The term “co-stimulatory signaling region (CSR)” refers to intracellular signaling domains from costimulatory protein receptors, such as CD28, 41 BB, and ICOS, that are able to enhance T-cell activation by T-cell receptors.

[0101] In some embodiments, the endodomain contains an SD or a CSR, but not both. In these embodiments, an immune effector cell containing the disclosed CAR is only activated if another CAR (or a T-cell receptor) containing the missing domain also binds its respective antigen.

[0102] Additional CAR constructs are described, for example, in Fresnak AD, et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81 , which is incorporated by reference in its entirety for the teaching of these CAR models.

[0103] For example, the CAR can be a TRUCK, Universal CAR, Self-driving CAR, Armored CAR, Self-destruct CAR, Conditional CAR, Marked CAR, TenCAR, Dual CAR, or sCAR.

[0104] TRUCKS (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T cell activation. Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.

[0105] Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0106] Self-driving CARs co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing tumor homing.

[0107] CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD 1 )), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.

[0108] A self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small-molecule dimerizer.

[0109] A conditional CAR T cell is by default unresponsive, or switched ‘off’, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1and signal 2, thereby activating the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.

[0110] Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects.

[0111] A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular co-stimulatory domain(s) and a CD3^ domain. TanCAR T cell activation is achieved only when target cells co-express both targets.

[0112] A dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3 domain and the other CAR includes only the costimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.

[0113] A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0114] The antigen recognition domain of the disclosed CAR is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.

[0115] The endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Therefore, the endodomain may comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co-receptors. While usually the entire intracellular signaling domain can be employed, 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 truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.

[0116] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, CD3^, CD36, CD3y, CD3E, CD32 (Fc gamma Rlla), DAP10, DAP12, CD79a, CD79b, FcyRly, FcyRllly, FcsRIp (FCERIB), and FCERIY (FCERIG).

[0117] In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3^) (TCR zeta, GenBank accession no. BAG36664.1). T-cell surface glycoprotein CD3 zeta (CD3^) chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.

[0118] First-generation CARs typically had the intracellular domain from the CD3^ chain, which is the primary transmitter of signals from endogenous TCRs. Second- generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, third-generation CARs combine multiple signaling domains to further augment potency. T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumoreradicating efficiency independent of costimulatory receptor / ligand interaction (Imai C, et al. Leukemia 2004 18:676-84; Maher J, et al. Nat Biotechnol 2002 20:70-5).

[0119] For example, the endodomain of the CAR can be designed to comprise the CD3^ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3^ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1 BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Thus, while the CAR is exemplified primarily with CD28 as the co-stimulatory signaling element, other costimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0120] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety (e.g., anti-CD30 scFv) and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from anysuitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0121] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1 BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11C, ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1 , CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1 , CD100 (SEMA4D) , SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1 , CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, and PAG / Cbp. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.

[0122] In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.

[0123] In some embodiments, the CAR is a multi-chain CAR, as described in WO2015 / 039523, which is incorporated by reference for this teaching. A multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction. For example, the multi-chain CAR can comprise a part of an FCERI alpha chain and a part of an FCERI beta chain such that the FCERI chains spontaneously dimerize together to form a CAR.

[0124] In some embodiments, the anti-CD30 binding agent is single chain variable fragment (scFv) antibody. The affinity / specificity of an anti-CD30 scFv is driven in large part by specific sequences within complementarity determining regions (CDRs) in the heavy (VH) and light (L) chain. Each VHand VLsequence will have three CDRs (CDR1 , CDR2, CDR3).

[0125] In some embodiments, the anti-CD30 binding agent is derived from natural antibodies, such as monoclonal antibodies. In some cases, the antibody is human. In some cases, the antibody has undergone an alteration to render it less immunogenic when administered to humans. For example, the alteration comprises one or more techniques selected from the group consisting of chimerization, humanization, CDR-grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequence.

[0126] Also disclosed are bi-specific CARs that target CD30 and at least one additional tumor antigen. Also disclosed are CARs designed to work only in conjunction with another CAR that binds a different antigen, such as a tumor antigen. For example, in these embodiments, the endodomain of the disclosed CAR can contain only an signaling domain (SD) or a co-stimulatory signaling region (CSR), but not both. The second CAR (or endogenous T-cell) provides the missing signal if it is activated. For example, if the disclosed CAR contains an SD but not a CSR, then the immune effector cell containing this CAR is only activated if another CAR (or T-cell) containing a CSR binds its respective antigen. Likewise, if the disclosed CAR contains a CSR but not a SD, then the immune effector cell containing this CAR is only activated if another CAR (or T-cell) containing an SD binds its respective antigen.

[0127] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The additional antigen binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the additional antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma- associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1 , MUC1 , BCMA, bcr-abl, HER2, p-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1 , ADRB3, thyroglobulin, EphA2, RAGE-1 , RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1 , fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6- AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1 , RUI, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1 , LAGE- la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1 , VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1 , prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1 , MAD-CT-2, MelanA / MART 1 , XAGE1 , ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1 , ephnnB2, CD20, CD22,CD24, CD30, TIM3, CD38, CD44v6, CD97, CD171 , CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61 , folate receptor (FRa), folate receptor beta, R0R1 , Flt3, TAG72, TN Ag, Tie 2, TEM1 , TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvlll, IL-13Ra, CD123, CD19, TIM3, BCMA, GD2, CLL-1 , CA-IX, MUCI, HER2, and any combination thereof.

[0128] Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY- ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1 , PSA, CA 19-9, CA 72-4, CAM 17.1 , NuMa, K-ras, beta-Catenin, CDK4, Mum-1 , p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1 , CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7- Ag, MOV18, NB / 70K, NY-CO-1 , RCASI, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1 , EBMA-1 , BARF-1 , CS1 , CD319, HER1 , B7H6, L1CAM, IL6, and MET.Nucleic Acids and Vectors

[0129] Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed CD30-specific CARs that allow expression of the CD30-specific CARs in the disclosed immune effector cells.

[0130] Nucleic acid sequences encoding the disclosed CARs, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0131] Expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter, and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0132] The disclosed 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, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0133] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful 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, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vectors are lentiviral or retroviral vectors.

[0134] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.

[0135] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1 a (EF-1a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0136] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elementsfrequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.

[0137] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.

[0138] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0139] Methods of 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, e.g., mammalian, bacterial, yeast, or 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.

[0140] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0141] 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 inserting genes into mammalian, e.g., human cells.

[0142] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0143] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).Immune effector cells

[0144] Also disclosed are immune effector cells that are engineered to express the disclosed CARs (also referred to herein as “CAR-T cells.” These cells are preferably obtained from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immuneeffector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0145] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

[0146] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0147] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.

[0148] Cytotoxic T cells (Tc cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0149] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0150] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Treg cells have been described — naturally occurring Tregcells and adaptive Tregcells.

[0151] Natural killer T (NKT) cells bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d.

[0152] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T comprise are cytotoxic CD8+T lymphocytes. In some embodiments, the T cells comprise y6 T cells, which possess a distinct T-cell receptor (TCR) having one y chain and one 6 chain instead of a and p chains.

[0153] Epstein-Barr virus (EBV)-induced lymphoproliferative diseases (EBV-LPDs) are a significant cause of morbidity and mortality for recipients of allogeneic hematopoietic cell transplantation (HCT), particularly in those who have received certain T-cell reactive Abs to prevent or treat GVHD. Prophylaxis and treatment by the adoptive transfer of EBV-specific T cells and the subsequent long-term restoration of immunity against EBV-associated lymphoproliferation have provided positive outcomes in the management of this uniformly fatal complication of bone marrow transfer. Therefore, in some embodiments, the disclosed immune effector cells are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, this can involve isolating PBMCs from of an autologous or allogenic donor and enriching them for T cells by depletion of monocytes and NK cells. For example, the donor can be an EBV-seropositive donor. These T cells can then be stimulated with autologous EBV-seropositive or transformed lymphocytes. EBV antigens include latent membrane protein (LMP) and EBV nuclear antigen (EBNA) proteins, such as LMP-1 , LMP- 2A, and LMP-2B and EBNA-1 , EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. These methods are described, for example, in Barker et al., Blood 2010 1 16(23): 5045-49; Doubrovina, et al., Blood 2012 119(1 1):2644-56; Koehne, et al. Blood 2002 99(5): 1730-40; and Smith et al. Cancer Res 2012 72(5): 11 16-25, which are incorporated by reference for these teachings.Therapeutic Methods

[0154] Immune effector cells expressing the disclosed CARs can elicit an anti-tumor immune response against CD30-expressing cancer cells. The anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response. In addition, the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific to CD30.

[0155] Adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anti-cancer therapeutic. Following the collection of a patient’s immune effector cells, the cells may be genetically engineered to express the disclosed CD30-specific CARs, then infused back into the patient.

[0156] The disclosed CAR-modified immune effector cells may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2, IL-15, or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise 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 (e.g., aluminum hydroxide); and preservatives. Compositions for use in the disclosed methods are in some embodiments formulated for intravenous administration. Pharmaceutical compositions may be administered in any manner appropriate treat MM. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0157] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104to 109cells / kg body weight, such as 105to 106cells / kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0158] In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently re-draw blood (or have an apheresis performed), activate T cells therefrom according to the disclosed methods, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw / multiple reinfusion protocol may serve to select out certain populations of T cells.

[0159] The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by i.v. injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0160] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immunecells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[0161] The cancer of the disclosed methods can be any CD30-expressing cell in a subject undergoing unregulated growth, invasion, or metastasis. Cancers that express CD30 include prostate cancer, ovarian cancer, adenocarcinoma of the lung, breast cancer, endometrial cancer, gastric cancer, colon cancer, and pancreatic cancer. CD30 has also been found on Jurkat cells. In some aspects, the cancer is a gallbladder cancer, exocrine adenocarcinoma, or apocrine adenocarcinomas. In some cases, the cancer comprises myelodysplastic syndrome, acute myeloid leukemia, or bi-phenotypic leukemia.

[0162] In some aspects, the cancer can be any neoplasm or tumor for which radiotherapy is currently used. Alternatively, the cancer can be a neoplasm or tumor that is not sufficiently sensitive to radiotherapy using standard methods. Thus, the cancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat include lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and pancreatic cancer.

[0163] The disclosed CARs can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.

[0164] The disclosed CARs can be used in combination with a checkpoint inhibitor. The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T- lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1 , an inhibitory signal is transmitted into theT cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0165] Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0166] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds PDL1 , such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds PD1 , such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0167] The disclosed CARs can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapy: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells, without necessarily initiating an immune response in the patient, while active immunotherapy actively triggers an endogenous immune response. Passive strategies include the use of the monoclonal antibodies (mAbs) produced by B cells in response to a specific antigen. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens permitted the pharmaceutical development of mAbs that could specifically target tumor cells for destruction by the immune system. Thus far, mAbs have been the biggest success story for immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein that is highly expressed on the surface of B cell malignancies such as non-Hodgkin’s lymphoma (NHL). Rituximab is approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Anotherimportant mAb is trastuzumab (Herceptin; Genentech), which revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0168] Generating optimal “killer” CD8 T cell responses also requires T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including 0X40 (CD134) and 4-1 BB (CD137). 0X40 is of particular interest as treatment with an activating (agonist) anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors.

[0169] In some embodiments, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0170] In some embodiments, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.

[0171] In some embodiments, such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0172] In some embodiments, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0173] In some embodiments, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBI (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).

[0174] In some embodiments, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0175] Therefore, in some embodiments, a disclosed antibody is used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0176] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0177] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a cell cycle control / apoptosis regulator (or "regulating agent"). A cell cycle control / apoptosis regulator may include molecules that target and modulate cell cycle control / apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01 , KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055) . Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

[0178] In some embodiments, a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, an antiandrogene (such as flutaminde / eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone / provera, megestrol acepate / megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole / arimidex, aminoglutethimide / cytraden, exemestane) or a hormone inhibitor (such as octreotide / sandostatin).

[0179] In some embodiments, a therapeutic agent for use in combination with an CARs for treating the disorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0180] Combined administration, as described above, may be simultaneous, separate, or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

[0181] In some embodiments, the disclosed CARs is administered in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241 , gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131 , and indium-111.

[0182] In some embodiments, the disclosed CARs is administered in combination with surgery.

[0183] CAR-T cells may be designed in several ways that enhance tumor cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and prolong their therapeutic half-life. TRUCK (T-cells Redirected for Universal Cytokine Killing) T cells for example, possess a CAR but are also engineered to release cytokines such as IL- 12 that promote tumor killing. Because these cells are designed to release a molecular payload upon activation of the CAR once localized to the tumor environment, these CAR-T cells are sometimes also referred to as ‘armored CARs’. Several cytokines as cancer therapies are being investigated both pre-clinically and clinically, and may also prove useful when similarly incorporated into a TRUCK form of CAR-T therapy. Among these include IL-2, IL-3. IL-4, IL- 5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-a, IFN-y, TNF-a, TRAIL, FLT3 ligand, Lymphotactin, and TGF-p (Dranoff 2004). “Self-driving” or “homing” CAR-T cells are engineered to express a chemokine receptor in addition to their CAR. As certain chemokines can be upregulated in tumors, incorporation of a chemokine receptor aids in tumor trafficking to and infiltration by the adoptive T-cell, thereby enhancing both specificity and functionality of the CAR-T (Moon 2011). Universal CAR-T cells also possess a CAR, but are engineered such that they do not express endogenous TCR (T-cell receptor) or MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signaling repertoire of the adoptive T-cell therapy prevents graft-versus-host-disease and rejection, respectively. Armored CAR-T cells are additionally so named for their ability to evade tumor immunosuppression and tumor-induced CAR-T hypofunction. These particular CAR-Ts possess a CAR, and may be engineered to not express checkpoint inhibitors. Alternatively, these CAR-Ts can be co-administered with a monoclonal antibody (mAb) that blocks checkpoint signaling. Administration of an anti-PDL1 antibody significantly restored the killing ability of CAR TILs (tumor infiltrating lymphocytes). While PD1-PDL1 and CTLA-4- CD80 / CD86 signaling pathways have been investigated, it is possible to target other immune checkpoint signaling molecules in the design of an armored CAR-T including LAG-3, Tim-3, IDO-1 , 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatases (SHP1),ubiquitin-ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase) . Armored CAR-Ts may also be engineered to express proteins or receptors that protect them against or make them resistant to the effects of tumor-secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with the double negative form of the TGF- receptor are resistant to the immunosuppression by lymphoma secreted TGF-p. These transduced cells showed notably increased antitumor activity in vivo when compared to their control counterparts.

[0184] Tandem and dual CAR-T cells are unique in that they possess two distinct antigen binding domains. A tandem CAR contains two sequential antigen binding domains facing the extracellular environment connected to the intracellular costimulatory and stimulatory domains. A dual CAR is engineered such that one extracellular antigen binding domain is connected to the intracellular costimulatory domain and a second, distinct extracellular antigen binding domain is connected to the intracellular stimulatory domain. Because the stimulatory and costimulatory domains are split between two separate antigen binding domains, dual CARs are also referred to as “split CARs”. In both tandem and dual CAR designs, binding of both antigen binding domains is necessary to allow signaling of the CAR circuit in the T-cell. Because these two CAR designs have binding affinities for different, distinct antigens, they are also referred to as “bi-specific” CARs.

[0185] One primary concern with CAR-T cells as a form of “living therapeutic” is their manipulability in vivo and their potential immune-stimulating side effects. To better control CAR-T therapy and prevent against unwanted side effects, a variety of features have been engineered including off-switches, safety mechanisms, and conditional control mechanisms. Both self-destruct and marked / tagged CAR-T cells for example, are engineered to have an “off-switch” that promotes clearance of the CAR-expressing T-cell. A self-destruct CAR-T contains a CAR, but is also engineered to express a pro-apoptotic suicide gene or “elimination gene” inducible upon administration of an exogenous molecule. A variety of suicide genes may be employed for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC TAG, and truncated EGFR (endothelial growth factor receptor). HSK for example, will convert the prodrug ganciclovir (GCV) into GCV-triphosphate that incorporates itself into replicating DNA, ultimately leading to cell death. iCasp9 is a chimeric protein containing components of FK506-binding protein that binds the small molecule AP1903, leading to caspase 9 dimerization and apoptosis. A marked / tagged CAR-T cell however, is one that possesses a CAR but also is engineered to express a selection marker. Administration of a mAb against this selection marker will promote clearance of the CAR-T cell. Truncated EGFR is one such targetable antigen by the anti-EGFR mAb, and administration of cetuximab works to promotes elimination of the CAR-T cell. CARs created to have these features are also referred to as sCARs for ‘switchable CARs’, and RCARs for ‘regulatable CARs’. A “safetyCAR”, also known as an “inhibitory CAR” (iCAR), is engineered to express two antigen binding domains. One of these extracellular domains is directed against a tumor related antigen and bound to an intracellular costimulatory and stimulatory domain. The second extracellular antigen binding domain however is specific for normal tissue and bound to an intracellular checkpoint domain such as CTLA4, PD1 , or CD45. Incorporation of multiple intracellular inhibitory domains to the iCAR is also possible. Some inhibitory molecules that may provide these inhibitory domains include B7-H1 , B7-1 , CD160, PIH, 2B4, CEACAM (CEACAM-1 . CEACAM-3, and / or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1 , and TGF -R. In the presence of normal tissue, stimulation of this second antigen binding domain will work to inhibit the CAR. It should be noted that due to this dual antigen specificity, iCARs are also a form of bi-specific CAR-T cells. The safety CAR-T engineering enhances specificity of the CAR-T cell for tumor tissue, and is advantageous in situations where certain normal tissues may express very low levels of a tumor associated antigen that would lead to off target effects with a standard CAR (Morgan 2010). A conditional CAR-T cell expresses an extracellular antigen binding domain connected to an intracellular costimulatory domain and a separate, intracellular costimulator. The costimulatory and stimulatory domain sequences are engineered in such a way that upon administration of an exogenous molecule the resultant proteins will come together intracellularly to complete the CAR circuit. In this way, CAR-T activation can be modulated, and possibly even ‘fine-tuned’ or personalized to a specific patient. Similar to a dual CAR design, the stimulatory and costimulatory domains are physically separated when inactive in the conditional CAR; for this reason these too are also referred to as a “split CAR”.

[0186] In some embodiments, two or more of these engineered features may be combined to create an enhanced, multifunctional CAR-T. For example, it is possible to create a CAR-T cell with either dual- or conditional- CAR design that also releases cytokines like a TRUCK. In some embodiments, a dual-conditional CAR-T cell could be made such that it expresses two CARs with two separate antigen binding domains against two distinct cancer antigens, each bound to their respective costimulatory domains. The costimulatory domain would only become functional with the stimulatory domain after the activating molecule is administered. For this CAR-T cell to be effective the cancer must express both cancer antigens and the activating molecule must be administered to the patient; this design thereby incorporating features of both dual and conditional CAR-T cells.

[0187] Several different methods for CAR expression may be used including retroviral transduction (including y-retroviral), lentiviral transduction, transposon / transposases (Sleeping Beauty and PiggyBac systems), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) has become of increasing importance with respect to the possibility forengineering CAR-T cells as well. CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator like effector nuclease) systems are three potential methods through which CAR-T cells may be generated.

[0188] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1:

[0189] Figure 1 shows topographical epitopes (Ep) of T105 and T25 on CD30 molecules. T105 recognizes the stalk region of Ep2A (from amino acid residues 107-153) proximal to the cleavage site (marked by scissors symbol) of the CD30 extracellular domain. This region remains membrane bound once the distal portion of CD30 is shed as soluble CD30. T25 recognizes Ep4 distal to the cleavage site of the CD30 extracellular domain. This region is shed as a soluble CD30 molecule.

[0190] Figure 2 shows humanization of T105 heavy (SEQ ID NO: 13) and light chain (SEQ ID NO:14) elements yields OSU-185 heavy (SEQ ID NO:1) and light (SEQ ID NO:2) chains. An antibody humanization machine learning model from Observed Antibody Space (OAS) was used to optimize humanization. Multiple iterations of mutations were tested, including 19 different mutations at all 83 positions of the heavy chain (VH) and light chain (VL) (framework) regions of the germline T105 sequence. Site-directed mutations on heavy and light chain’s amino acid residues were targeted outside complementarity determining regions (CDR’s) 1-3 to preserve the antibody binding domains. The resultant top 5 germline sequences were used for testing, including IGHV2-5*08 / IGHJ6*04, IGHV2-5*09 / IGHJ6*01 , IGHV2-5*02 / IGHJ2*01, IGHV2-5*05 / IGHJ3*01 , and IGHV2-5*01 / IGHJ4*01 for the heavy chain and IGKV1-5*01 / IGKJ2*01 , IGKV1-5*03 / IGKJ2*02, IGKV1-9*01 / IGKJ5*01, IGKV1- 12*01 / IGKJ1*01 , and IGKV1-27*01 / IGKJ2*03 forthe light chain. These humanized sequences were compared to T105 heavy and light chain sequences and subjected to a deimmunization protocol. This protocol allowed for identification and substitution of the amino acid residues in a 15mer window size and 10mer overlap to alter their characteristics from immunogenic to non-immunogenic for each selected peptide presented by a 26 reference allele set for major histocompatibility complex class II molecules. Consideration on the effect of the substitute on the neighboring peptides on the VH and VL chains was made to counter any potential negative effect on the antibody folding and binding affinity.

[0191] Figure 3 shows substantial increase in OASis identity and germline content with antibody humanization reveals increase in the humanness score of OSU-185. Percentage of OASis identity and germline content for original murine (T 105) and humanized(OSU-185) antibodies are shown. OASis (Observed Antibody Space identity search) is a novel antibody humanness score based on exact 9-mer peptide search in the Observed Antibody Space (OAS) database and provides an interpretable and granular humanness report with adjustable stringency on antibody protein sequence. Site-directed mutations on heavy and light chain’s amino acid residues outside complementarity determining regions (CDRs) preserve the antibody binding domains CDR1-3.

[0192] Figures 4Ato 4B show pairwise protein sequence alignment between T105 and final humanized OSU-185 sequence. Figure 4A shows pairwise protein sequence alignment on the heavy chains of T105 and OSU-185 performed Smith-Waterman alignment algorithm. 110 out of 121 amino acid residues (90.9%) identity and 114 out of 121 amino acid residues (94.2%) are identical and similar, respectively. Figure 4B shows pairwise protein sequence alignment on the light chains of T105 and OSU-185. 97 out of 107 amino acid residues (90.7%) identity and 99 out of 107 amino acid residues (92.5%) are identical and similar, respectively.

[0193] Figures 5A and 5B show design of a CD30 CAR lentiviral vectors. Figures 5A and 5B show OSU-184 (Fig. 5A) and OSU-185 (FIG. 5B) CD30 scFV with CD8a hinge region, CD8a transmembrane domain (TM); signaling domain of 4-1 BB molecule (4-1 BB); and CD3 zeta chain (CD3^).

[0194] Figure 6 shows expression of CD30 on classical Hodgkin’s Lymphoma (cHL) and control cell lines. CD30 surface expression of unstained and stained cell lines analyzed by flow cytometry. cHL cell lines included KM-H2, HDLM2, L428, L540, and L-1236 and in majority showed similar degrees of CD30 positivity. An example CD30 negative cell line (Mino - mantle cell lymphoma) is also shown. Cells were washed once with PBS, blocked with Trustain human Fc blocker (BioLegend), stained with antibodies for 20 minutes at room temperature and analyzed with a LSRII flow cytometer (BD Biosciences, CA, USA).

[0195] Figure 7 shows efficient transduction with the OSU-184 and OSU-185 CD30 CAR vectors. Naive T cells were isolated using pan naive T cell isolation kit (Miltenyi) according to the manufacturer’s instructions. The cells were primed overnight with IL-15, -7 and -21 at 25ng / mL and human T cell TransAct and subjected to lentiviral transduction with the lentivirus carrying the OSU-184 or -185 CAR constructs. The transduction efficiencies of untransduced naive T cells, OSU-184 and OSU-185 on Day 3 and 6 post transduction were performed by flow cytometry. GS4 linker antibody was used to detect CAR+ T-cells.

[0196] Figure 8 shows cytotoxicity analysis of OSU-184 and OSU-185 CD30 CAR-T cells against 2 classical Hodgkin lymphoma (cHL) cell lines: L-428 and L-540. Untransduced T cells (UTT), OSU-184, or OSU-185 (CD30 CAR-T cells were co-cocultured with target cells at E:T ratio of 5:1 for 24 hours before the specific lysis was determined. Two cell lines with known low / negative CD30 expression were used as controls and included DC9 (EBVinfected B cell lymphoma cell line) and Mino (mantle cell lymphoma cell line). Ns: not significant; ** p<0.01 ; ****p<0.0001 .

[0197] Table 1: Humanness score of 7 V-gene type of T105 heavy chain. A random forest (RF) machine learning model was used to determine that the heavy chain of T105 is non-human as determined by the humanness score threshold (Marks C et.al. 2021). The humanness score was produced by the RF model based on similarities and differences between human V genes and the tested non-human variable domain sequences. The humanness score describes how typical an antibody sequence is of the human repertoire and provides the ability to distinguish human antibodies from those of other species. Humanness scores derived from this model are highly negatively correlated to observed immunogenicity (i.e. the higher the score, the lower the immunogenicity of the antibody).Table 1 : Humanness score of 7 V-gene type of T105 heavy chain.Score Threshold Human?HV1 0 0.725 NotHV2 0 0.835 NotHV3 0 0.575 NotHV4 0 0.565 NotHV5 0 0.52 NotHV6 0 0.93 NotHV7 0 0.72 NotTable 2: Humanness score of 6 V-gene type of T105 light chain. A random forest(RF) machine learning model was used to determine that the light chain of T105 is nonhuman as determined by the humanness score threshold (Marks C et.al. 2021).Table 2: Humanness score of 6 V-gene type of T105 light chain.Score Threshold Human?KV1 0 0.72 NotKV2 0 0.72 NotKV3 0 0.72 NotKV4 0 0.655 NotKV5 0 0.815 NotKV6 0 0.475 NotExample 2:

[0198] To test the immunogenicity of the humanized OSU-184 and OSU-185 CAR constructs, the following immunogenicity assay was performed. Separate batches of CAR-T cells were generated using OSU-184 and OSU-185 and non-humanized murine T25 andT105 single chain variable fragment controls. Cell lysates from each set of CAR-T cells were prepared, fragmented and pulsed with human peripheral blood mononuclear cells (PBMCs) for 6 days. T cell activation induced by the peptide antigen presentation in each cell culture was determined by CD3 and CD25 expression using flow cytometry. The mean fluorescence intensity of induced CD25 (which correlates to activation level) in OSU-184 pulsed PBMCs was 24 compared to 30 in T25 (murine) pulsed cells which was a 20% decrease (Fig. 9). Additionally, induced CD25 expression in OSU-185 pulsed cels was 4 compared to 86 in T105 (murine) which was a 95.3% decrease (Fig. 9). These results indicate the humanization process resulted in less a less immunogenic OSU-184 and 185 CART construct compared their murine counterparts.Example 3:

[0199] We investigated the in-vivo activity of T25 and OSU-185 CD30 CAR-T cells in a preclinical cHL L-540 cell derived xenograft mouse model using NOD-SCIDvc / mice. L-540 cells were genetically modified to express luciferase, which allowed for real-time monitoring of lymphoma cell growth using IVIS imaging. Mice were engrafted with luciferase expressing L-540 cells subcutaneously near the flank and then were randomized to receive PBS (negative control), UTT (untransduced T cells), T25 or OSU-185 CAR-T cells. Mice treated with T25 and OSU-185 CD30 CAR-T cells had lower average tumor burden as detected by the IVIS imaging compared to controls (Fig. 10). Tumors in mice treated with OSU-185 CD30 CAR-T cells had the smallest average sizes among all 4 groups at Day 14 (Fig. 10). The reduction in tumor burden in OSU-185 CD30 CAR-T treated mice indicates the humanization process allowed for maintained efficacy of OSU-185 CD30 CAR-T cells in a pre-clinical in-vivo cHL model.

[0200] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0201] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1 . A fully humanized single chain variable fragment (scFv) which targets the proximal portion of the CD30 molecule, wherein the scFv comprises a variable heavy (VH) domain having CDR1 , CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1 , CDR2 and CDR3 sequences, wherein the VHdomain comprises the amino acid sequence SEQ ID NO:1 , or a variant thereof having at least 95% identity to SEQ ID NO:1 , 15, 16, or 17; wherein the VLdomain comprises the amino acid sequence SEQ ID NO:2, or a variant thereof having at least 95% identity to SEQ ID NO:2, 18, 19, or 20; and wherein the CDR1 sequence of the V domain comprises the amino acid sequence SEQ ID NO:3; the CDR2 sequence of the VHdomain comprises the amino acid sequence SEQ ID NO:4; the CDR3 sequence of the VHdomain comprises the amino acid sequence SEQ ID NO:5; the CDR1 sequence of the VLcomprises the amino acid sequence SEQ ID NO:6; the CDR2 sequence of the VL domain comprises the amino acid sequence SEQ ID NO:7; and the CDR3 sequence of the VL domain comprises the amino acid sequence SEQ ID NO:8.

2. The scFv of claim 1 , wherein the H domain comprises the amino acid sequence SEQ ID NO:1 and wherein the L domain comprises the amino acid sequence SEQ ID NO:2.

3. The scFv of claim 1 , comprising the amino acid sequence SEQ ID NQ:10, or a variant thereof having at least 95% identity to SEQ ID NO: 10.

4. The scFv of claim 1 , wherein the VHdomain comprises the amino acid sequence SEQ ID NO:15, and wherein the VLdomain comprises the amino acid sequence SEQ ID NO:18.

5. The scFv of claim 1 , comprising the amino acid sequence SEQ ID NO:21 , or a variant thereof having at least 95% identity to SEQ ID NO:21.

6. The scFv of claim 1 , wherein the VHdomain comprises the amino acid sequence SEQ ID NO: 16, and wherein the VLdomain comprises the amino acid sequence SEQ ID NO:19.

7. The scFv of claim 1 , comprising the amino acid sequence SEQ ID NO:22, or a variant thereof having at least 95% identity to SEQ ID NO:22.

8. The scFv of claim 1 , wherein the VHdomain comprises the amino acid sequence SEQ ID NO:17, and wherein the VLdomain comprises the amino acid sequence SEQ ID NO:20.

9. The scFv of claim 1 , comprising the amino acid sequence SEQ ID NO:23, or a variant thereof having at least 95% identity to SEQ ID NO:23.

10. A chimeric antigen receptor (CAR) polypeptide, comprising a CD30 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co-stimulatory signaling region, wherein the CD30 antigen binding domain comprises the fully humanized scFv of claim 1.11 . The CAR polypeptide of claim 10, wherein the costimulatory signaling region comprises the cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1 BB, 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

12. The CAR polypeptide of claim 10 or 11 , wherein the intracellular signaling domain comprises a CD3 zeta (CD3< signaling domain.

13. The CAR polypeptide of claim 10, comprising the amino acid sequence SEQ ID NO:12.

14. A method of providing an anti-cancer immunity in a subject with a CD30-expressing cancer, the method comprising administering to the subject an effective amount of an immune effector cell genetically modified to express the CAR polypeptide of any one of claims 4 to 7, thereby providing an anti-tumor immunity in the mammal.

15. The method of claim 14, wherein the CD30-expressing cancer comprises Hodgkin’s Lymphoma, Non-Hodgkin’s Lymphoma.

16. The method of claim 14, wherein the CD30-expressing cancer comprises Classic Hodgkin’s Lymphoma (cHL).

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