(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`
`12 August 2010 (12.08.2010)
`
`(10) International Publication Number
`WO 2010/089412 Al
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH,CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID,IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM,PE, PG, PH,PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(51) International Patent Classification:
`CO7K 14/705 (2006.01)
`sD IT
`ional Application Number:
`(21)
`International
`Application Number:
`
`PCT/EP2010/051565
`
`(22) International Filing Date:
`
`9 February 2010 (09.02.2010)
`
`English
`English
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`61/150,934
`
`9 February 2009 (09.02.2009)
`
`us (84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`except US):
`(for all designated States
`(71) Applicant
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`MUENCHEN
`HELMHOLTZ
`ZENTRUM
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK,EE,
`DEUTSCHES
`FORSCHUNGSZENTRUM FUER
`ES, FI, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU, LV,
`GESUNDHEIT UND UMWEL'T(GMBH) [DE/DE];
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM,
`Ingolstaedter Landstrasse 1, 85764 Neuherberg (DE).
`TR), OAPI (BF, BJ, CF, CG, CL, CM, GA, GN, GQ, GW,
`(72) Inventors; and
`ML, MR, NE, SN, TD, TG).
`(75) Inventors/Applicants (for US only): SCHENDEL, Do-
`lores J.
`[US/DE]; Hans-Sachs-Sirasse
`12/[TV, 80469 Published:
`Muenchen (DE). WILDE, Susanne [DE/DE]; Zcppclin-
`strasse 59, 81669 Muenchen (DE). FRANKENBERG- “international search report(Art. 21(3))
`ER, Bernhard [DE/DE]; Kirchbachweg
`7,
`81479 — before the expiration ofthe time limit for amending the
`Muenchen (DE). UCKERT, Wolfgang [DE/DE]; Strasse
`claims and to be republished in the event of receipt of
`45 Nr. 28, 13125 Berlin (DE).
`amendments (Rule 48.2(h))
`
`(74) Agent: SANDMANN, Wolfgang; Friedrichstrasse 31,
`80801 Miinchen (DE).
`
`(54) Title: REPERTOIRE OF ALLO-RESTRICTED PEPTIDE-SPECIFIC T CELL RECEPTOR SEQUENCES AND USE
`THEREOF
`
`(57) Abstract: The present invention is directed to a kit-of-parts or composition containing nucleic acid sequences coding for
`high-avidity, allo-restricted TCR, wherein the TCR are independently directed against the tyrosinase antigen, the melan-A antigen
`and the survivin antigen. The invention is further directed to a kit-of-parts or composition containing at least three groups oftrans-
`genic lymphocytes transformed with vectors coding for TCR against said antigens. Furthermore, the present invention provides a
`pharmaceutical composition and its use in the treatment of diseases involving malignant cells expressing said tumor-associated
`antigens. The invention further relates to a nucleic acid molecule coding for a TCR that recognizes the survivin antigen, a TCR
`encoded thereby and a T cell expressing said TCR. Further, the invention discloses a vector, a cell and a pharmaceutical composi-
`tion encoding/containing same andtheir use in the treatment of diseases involving malignant cells expressing survivin.
`
`
`
`wo2010/089412AIMITINMININUATNINNAAIATA
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`Repertoire of allo-restricted peptide-specific T cell receptor sequences and use thereof
`
`FIELD OF THE INVENTION
`
`The present invention is directed to a kit-of-parts or composition containing nucleic acid
`
`sequences coding for high-avidity, allo-restricted TCR, wherein the TCR are independently
`
`directed against the tyrosinase antigen, the melan-A antigen and the survivin antigen. The
`
`invention is further directed to a kit-of-parts or composition containing at least three groups of
`transgenic lymphocytes transformed. with vectors coding for TCR against said antigens.
`Furthermore, the present invention provides a pharmaceutical composition and its use in the
`
`treatment of diseases involving malignant cells expressing said tumor-associated antigens.
`
`The invention further relates to a nucleic acid molecule coding for a TCR that recognizes the
`
`survivin antigen, a TCR encoded thereby and a T cell expressing said TCR. Further, the
`
`invention discloses a vector, a cell and a pharmaceutical composition encoding/containing
`
`sameand their use in the treatment of diseases involving malignant cells expressing survivin.
`
`BACKGROUND OF THE INVENTION
`
`T cell responses against tumors are often directed against self-MHC molecules presenting
`
`peptides derived from over-expressed self-proteins. In general, T cells with high avidity for
`self-peptide/self-MHCligands are eliminated by negative selection to prevent autoimmunity.
`
`The TCRaffinity of remaining T cells specific for self-ligands is normally low, however high-
`
`avidity T cells are needed to effectively eradicate tumors. Because negative selection is
`
`limited to self-MHC molecules, T cells that recognize allogeneic MHC molecules have not
`
`undergone negative selection. Thus, if peptides are presented by allogeneic MHC molecules,
`it is feasible to obtain high-avidity T cells specific for common tumor-associated ligands
`derived from over-expressed self-proteins. T cells that recognize allogeneic MHC molecules
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`2
`
`irrespective of a specific peptide can be distinguished in vitro from allo-restricted peptide-
`specific T cells at the clonal level and excluded.
`
`Significant tumor regression can occur following adoptive transfer of T cells with anti-tumor
`
`specificity. However, patient-derived T cells may have sub-optimal activity. Furthermore, T
`
`cells with appropriate specificity and function for effective tumor eradication are often not
`
`available for patients with rapidly progressing tumors. Therefore, there is current interest in
`
`using pre-characterized TCR genes to create designer lymphocytes for adoptive cell therapies.
`Expression of TCR-transgenes in activated lymphocytes can imbue recipient Lymphocytes
`with anti-tumor activities comparable to the original T cells (Morris et al. Blood Rev (2006)
`20, 61-69; Schumacher et al., Nat. Rev. Immunol. (2002) 2, 512-519). Moreover, some
`transgenic TCR can displace endogenous TCR sequences, yielding lymphocytes that express
`monoclonal TCR.
`
`Thefirst clinical trials using adoptive transfer of TCR-transgenic T cells in melanomapatients
`achieved clinical disease-free status in 2 of 17 patients with rapidly progressing disease
`(Morgan et al. Science (2006) 314, 126-129). Higher rates of clinical efficacy were obtained
`in patients receiving TCR transgenic lymphocytes transduced with a TCR of higheraffinity
`but some undesired responses were noted against normal tissues. These results demonstrated
`the therapeutic potential of this approach howeverthey also revealed the need to evaluate a
`
`variety of TCR sequences that recognize the same ligand but have different affinities in order
`
`to identify the most suitable TCR sequences for clinical development that can be used to
`achieve optimal elimination of tumor cells while showing the lowest undesired activity
`directed against normal, non-malignanttissues.
`
`A number of T cell clones with specificity for various tumor-associated antigens have been
`reported over the yéars. Most of these TCR are restricted by self-MHC molecules. Further,
`
`available TCR are often of low-avidity. Multiple TCR with good capacity to recognize tumor
`cells via different tumor-associated antigens (TAA)are often lacking.
`
`In the prior art, several scientific and patent documents are existing which describe TCR that
`are able to recognise and bind specific antigens, for example tyrosinase. Visseren et al. (Int. J.
`Cancer (1997) 72, 1122-1128) describe the affinity and specificity of several tyrosinasc-
`specific TCR and suggest to use these TCR as a specific treatment of melanomapatients.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`Roszkowski et al.
`
`(JJ. Immunol. (2003) 170, 2582-2589 and Cancer Res. (2005) 65, 1570-
`
`1576) are likewise characterising tyrosinase-specific TCR.
`
`US 5,906,936 is directed to cytotoxic T-cells which kill non-MHC-restricted target cells
`independent of MHC-restriction and not to T-cells, which utilize specific TCR sequencesthat
`
`recognize MHC-restricted ligands.
`
`W097/32603 is directed to a method for producing non-human TCR and TCR specific for
`
`human HLA-restricted tumor antigens. Furthermore, the TCR-nucleic acids and recombinant
`
`T-cells are described as well as the administration of TCR recombinant T-cells for the
`
`treatment of several diseases.
`
`W02007/065957 describes an effector T-cell transfected with an antigen specific TCR coding
`RNA wherein the transfected T-cell recognizes the antigen in a complex with the MHC-
`molecule and binds the same. As potential tumor antigens, MART-1 (melan-A), tyrosinase
`and survivin are named.
`
`WO0O2008/039818 discloses MART-1 and tyrosinase-specific TCR sequences and describes
`
`the enhancement of antigen recognition by substitution in the CDR2 region.
`
`The above prior art TCR sequences are all derived from autologous or xenogeneic, but not
`
`allogeneic, sources.
`
`from tumor-infiltrating
`For example, TCR sequences are from peripheral blood or
`lymphocytes of HLA-A2-positive melanoma patients. This means that all these TCR are
`
`HLA-A2self-restricted TCRs, or, are HLA-DP4self-restricted, NY-ESO-1 specific, both
`derived from autologous sources. As an alternative, as disclosed in WO97/32603, the TCR is
`
`_ derived from an HLA-A2 transgenic mouse and, therefore, the sequence is xenogeneicin this
`case.
`
`However, the available prior art documents do not show TCR sequences, which are allo-
`restricted and specific for the survivin, tyrosinase and melan A antigens.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`Thus, there is still an important need to find means to generate T cells that bear TCR with
`
`high functional avidity that have the capacity to recognize specific ligands on tumorcells.
`
`Immuneselection of tumorcells poses a severe problem in TCR-based therapies. Tumors tend
`to be genetically unstable and maylose their antigens by mutation. This instability may Icad
`to the generation of antigen-loss variants which are able to escape the immuneresponse.
`Therefore, if tumor cells are attacked by T cells recognizing only one single TAA specificity,
`this might lead to a reduced or even absent success of therapy due to outgrowth of tumorcells
`lacking expression of the specific TAA.
`
`there is a further need existing to provide a clinical approach to effectively
`Therefore,
`minimize immuneselection of tumorcells and to provide a broad and specific attack on tumor
`cells.
`
`SUMMARY OF THE INVENTION
`
`Therefore, it is an object of the present invention to provide a TCR-based approach in orderto
`overcome the drawbacks of the prior art therapies,
`in particular to effectively minimize
`immune selection of tumorcells. It is a further object of the invention to provide a repertoire
`of TCR which can be effectively used in the treatment of diseases involving malignantcells
`expressing tyrosinase and/or melan-A and/or survivin, preferably melanomas, gliomas,
`glioblastomas, and/or rare tumors of ectodermalorigin, the like to provide mixtures of TCR-
`transgenic lymphocytes to target tumors via several different MHC-peptide ligands in order to
`avoid immuneselection of tumorcells that lack expression of a specific TAA.It is a further
`object of the present invention to provide TCR or functional parts thereof, such as CDR3
`regions, which show highaffinity against the survivin antigen.It is a still further object of the
`invention to provide pharmaceutical compositions for use in adoptive cell therapy which
`allow an effective treatmentof diseases involving malignantcells expressing survivin.
`
`These objects are solved by the subject-matter of the independent claims. Preferred
`embodiments are indicated in the dependentclaims.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`It is a great advantage to administer mixtures of TCR-transgenic specific T cells to patients to
`target their tumors via several different MHC-peptide ligands in order to avoid immune
`selection of tumorcells that lack TAA expression if they are attacked by T cells with only a
`single specificity.
`
`The inventors generated high-avidity, allo-restricted peptide-specific T cells that provide
`suitable sources of TCR sequences for selection of TCR that can be developed for clinical
`application. Furthermore,
`the inventors have generated a series of T cell clones and
`
`demonstrated their high-avidity and tumor-specificity for three distinct melanoma-associated
`
`antigens. In addition, one of the antigens for which they have generated a repertoire of TCR
`
`sequences, namely survivin, is broadly expressed in a variety of tumors and therefore, these
`
`sequences can also be used for treatment of tumors other than melanoma.
`
`The use of repertoires of TCR with different specificities does not only provide a broader
`basis of an attack of tumor cells, helping to avoid immune selection of TAA loss variants, but
`will also allow patients to be treated if their tumors naturally fail to express any one of the
`individual TAA that are targeted by the TCR. Thereby, future adoptive T cell therapies can be
`realized for more patients by employing these TCR sequences to develop “off the shelf”
`
`reagents for transduction of patient-derived lymphocytes.
`
`The combination of TCR used in the present invention, i.e. TCR directed against the survivin,
`
`is particularly effective in vivo in minimizing
`tyrosinase and optionally melan A antigen,
`immune selection of tumorcells and in defeating malignancies. In other words, also in case of
`
`immuneselection, there is still a high probability that the tumorto be attacked still expresses
`at least one of the named TAA and thus can beeffectively recognized and defeated. This is in
`contrast to prior art approaches, where tumor cells are attacked by T cells recognizing only
`one single TAA specificity, potentially leading to a reduced or even absent success of therapy
`due to outgrowth of tumorcells lacking expression of the specific TAA.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`According to a first aspect, the invention provides a kit-of-parts or composition comprising:
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`a group of vectors contaming nucleic acid sequences coding for high-avidity,
`a)
`allo-restricted TCR, wherein the TCR are directed against the tyrosinase antigen;
`b)
`a group of vectors containing nucleic acid sequences coding for high-avidity,
`allo-restricted TCR, wherein the TCR are directed against the melan-A antigen; and
`c)
`a group of vectors containing nucleic acid sequences coding for high-avidity,
`allo-restricted TCR, wherein the TCR are directed against the survivin antigen.
`
`As used herein, the term "kit-of-parts” shall encompass an entity of physically separated
`components, which are intended for individual usc, but in functional relation to each other.
`This means that the individual parts of the kit are provided for simultaneous or subsequent
`administration. If all components (or groups) are provided in mixed form, they are defined
`herein as a "composition" and not as a kit-of-parts.
`
`In an embodiment, the vector used in the kit-of-parts or composition is a plasmid, shuttle
`vector, phagemide, cosmid, expression vector, retroviral vector, adenoviral vector or particle.
`In the context of the present invention, a “vector” shall mean a nucleic acid molecule as
`
`introduced into a host cell, thereby producing a transformed host cell. A vector may include
`nucleic acid sequences that permitit to replicate in a host cell, such as an origin ofreplication.
`A vector may also include one or more selectable marker genes and other genetic elements
`known to those of ordinary skill in the art. A vector preferably is an expression vector that
`includes a nucleic acid according to the present invention operably linked to sequences
`allowing for the expression of said nucleic acid.
`
`In a preferred embodiment,the kit-of-parts or composition contains the following selection of
`vectors:
`
`The vectors of group a) are comprising at least one CDR3 sequence according to SEQ ID NO:
`1-10, or at least one nucleic acid sequence coding for the amino acid sequence of SEQ ID
`NO: 29-38 and/or
`
`the vectors of group b) are comprising at least one CDR3 sequence according to SEQ ID NO:
`11-20 or at least one nucleic acid sequence coding for the amino acid sequence of SEQ ID
`NO: 39-48, and/or
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`the vectors of group c) are comprising at least one CDR3 sequence according to SEQ ID NO:
`21-28 or at least one nucleic acid sequence coding for the amino acid sequence of SEQ ID
`NO: 49-56.
`
`It is noted that within each group, a ranking of the most promising sequences is existing,
`being from the most to the less preferred sequence:
`
`Directed against the tyrosinase antigen: CDR3 sequence according to SEQ ID NO: 1, 2, 8, 9,
`
`10, 3, 4, 5, 6, 7 or the nucleic acid sequence coding for the amino acid sequence of SEQ ID
`NO: 29, 30, 36, 37, 38, 31, 32, 33, 34, 35.
`
`Directed against the melan-A antigen: CDR3 sequence according to SEQ ID NO: 19, 20, 15,
`16, 17, 18, 11, 12, 13, 14 of the nucleic acid sequence coding for the amino acid sequence of
`SEQ ID NO: 47,48, 43, 44, 45, 46, 39, 40, 41, 42.
`
`Directed against the survivin antigen: CDR3 sequence according to SEQ ID NO: 27, 28, 23,
`
`24, 25, 26, 21, 22 or the nucleic acid sequence coding for the amino acid sequence of SEQ ID
`
`NO: 55, 56, 51, 52, 53, 54, 49, 50.
`
`It is further noted that, in the present invention, SEQ ID NOs defining the alpha and beta
`chains of a precise TCR are not grouped separately. Althoughit is contemplated that all alpha
`chain sequences may be combined with all beta chain sequences(if directed against the same
`antigen), it is preferred that the alpha and the beta chain sequences derived from the same
`clone are used in combination. For example, a preferred TCR against the survivin antigen may
`comprise SEQ ID NO: 27 for the alpha chain sequence and SEQ ID NO: 28for the beta chain
`
`sequence (both derived from the sameclone,i.e. SW-Surv-72).
`
`The invention further provides derivatives of said CDR3 sequences wherein the CDR3 region
`has been altered by one or more additions and/or deletions of an overall number of from 1-5
`
`amino acids, but not more than 1-3 contiguous amino acids and/or conservative substitutions
`
`of from 1-6 amino acids and wherein the tumor antigen recognizing characteristics are
`maintained or improved.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`This means, moreprecisely, that additions or deletions may be performedto an extent that 1-5
`amino acids are added or deleted in the CDR3 region. If more than one addition or deletion is
`
`performed, the overall number of added or deleted amino acids may not exceed 5 amino acids.
`
`Further, one single addition or deletion at one site may only be in the range of 1-3 amino
`
`acids, i.e. 1-3 contiguous amino acids, since the ligand binding capacity might be deteriorated
`
`by performing larger additions/deletions.
`
`In a further embodiment, the vectors are each comprising a nucleic acid molecule coding for
`the V(D)J regions of a TCR that recognizes the respective tumor antigen,
`the vectors
`
`comprising
`
`a) the nucleic acid sequence of SEQ ID NO: 57, 59, 61, 62, 64, or 65 coding for the o-chain
`
`and/or the nucleic acid sequence of SEQ ID NO: 58, 60, 63, or 66 coding for the B-chain of a
`
`TCRdirected against the tyrosinase antigen,
`b) the nucleic acid sequence of SEQ ID NO: 67, 69, 71, 73, or 75 coding for the o-chain
`and/or the nucleic acid sequence of SEQ ID NO: 68, 70, 72, 74, or 76 coding for the B-chain
`of said TCR directed against the melan-A antigen, and
`
`c) the nucleic acid sequence of SEQ ID NO: 77, 79, 81, or 83 coding for the a-chain and/or
`
`the nucleic acid sequence of SEQ ID NO:78, 80, 82, or 84 coding for the B-chain of said TCR
`directed against the survivin antigen,
`
`or a derivative of these sequences, coding for the a- or B-chain, wherein the chain has been
`altered by one or more additions or deletions of from 1-15 amino acids, the additions or
`
`deletions being outside the CDR3 region of each chain and/or by conservative substitutions of
`
`from 1-15 amino acids, wherein the tumor antigen recognizing characteristics are maintained
`
`or improved.
`
`Also here, a ranking of the most promising sequences is existing, being from the most to the
`
`less preferred sequence:
`
`Directed against the tyrosinase antigen: the nucleic acid sequence of SEQ ID NO:57, 59, 64,
`65, 61, 62, coding for the a-chain and/or the nucleic acid sequence of SEQ ID NO: 58, 60, 66,
`63 coding for the B-chain of a TCR directed against the tyrosinase antigen.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`Directed against the melan-A antigen: the nucleic acid sequence of SEQ ID NO: 75, 71, 73,
`
`67, 69 coding for the a-chain and/or the nucleic acid sequence of SEQ ID NO: 76, 72, 74,
`
`68, 70 coding for the B-chain of said TCR directed against the melan-A antigen.
`
`Directed against the survivin antigen: the nucleic acid sequence of SEQ ID NO: 83, 79, 81,
`and 77 coding for the a-chain and/or the nucleic acid sequence of SEQ ID NO:84, 80, 82, and
`78 coding for the B-chain of said TCR directed against the survivin antigen,
`
`The term "nucleic acid" as used hercin refers to a naturally-occurring nucleic acid that is not
`immediately contiguous with both of the sequences with which it is immediately contiguous
`(one on the 5' end and one onthe 3' end) in the naturally-occurring genomeof the cell from
`
`which it is derived. For example, a nucleic acid can be, without limitation, a recombinant
`
`DNA molecule of any length, provided one of the nucleic acid sequences normally found
`
`immediately flanking that recombinant DNA molecule in a naturally-occurring genomeis
`
`removed or absent. Thus, a nucleic acid includes, without limitation, a recombinant DNA that
`
`exists as a Separate molecule (e. g.,a cDNA or a genomic DNA fragment produced by PCR or
`restriction endonuclease treatment) independent of other sequences as well as recombinant
`DNAthat is incorporated into a vector, an autonomously replicating plasmid, a virus(e. g., a
`
`retrovirus, or adenovirus). In addition, an isolated nucleic acid can include a recombinant
`
`DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
`
`Furthermore, the term "nucleic acid" as used herein also includesartificially produced DNA
`
`or RNA sequences, such as those sequences generated by DNA or RNA synthesis based on in
`silico information.
`
`The invention is also directed to a kit-of-parts or composition comprising TCR, preferably
`soluble TCR, encoded by the above indicated nucleic acids and directed against the survivin,
`melan-A and tyrosinase antigens. These TCR may as an alternative be synthetic proteins.
`
`The nucleic acids of the invention can comprise natural nucleotides, modified nucleotides,
`
`analogues of nucleotides, or mixtures of the foregoing as long as they are capable of causing
`the expression of a polypeptide in vitro, and preferably, in a T cell. The nucleic acids of the
`
`invention are preferably RNA, and more preferably DNA.
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`10
`
`Furthermore, the present invention also comprises derivatives of the above described nucleic
`acid molecules, wherein, related to the above sequences, the sequence has been altered by
`additions, deletions and/or
`substitutions and wherein the tumor antigen recognizing
`characteristics are maintained or improved.
`
`Moreprecisely, such a derivative is coding for the a- or B-chain, wherein the chain has been
`
`altered by one or more additions or deletions of from 1-15 amino acids, the additions or
`
`deletions being outside the CDR3 region of each chain, and/or by conservative substitutions
`of from 1-15 amino acids. It is noted in this conncction that also the CDR3 region may be
`altered, but to a lesser extent. The definition of those amendments is indicated abovefor the
`
`derivatives of fragments coding for the CDR3 region.
`
`Useful changes in the overall nucleic acid sequence in particular are related to codon
`optimization and the addition of epitope tags, which will be explained in detail below. Such
`codon optimization can include optimization of expression levels, optimization of avidity for
`target cells, or both.
`
`In general, it should, however, be noted that the alterations should not diminish or alter the
`
`ability of the encoded polypeptide to form part of a TCR that recognizes tumor associated
`
`antigens in the context of an MHC molecule, but should facilitate destruction of a tumorcell,
`
`and preferably facilitate the regression of a tumor, or other cancerousstate.
`
`For example, alterations can be made which lead to conservative substitutions within the
`
`expressed amino acid sequence. These variations can be made in complementarity
`determining and non-complementarity determining regions of the amino acid sequenceofthe
`TCR chain that do not affect function. However, as noted above, additions and deletions
`
`should not be performed in the CDR3 region (for example an addition of epitope tags).
`
`The concept of "conservative amino acid substitutions" is understood by the skilled artisan,
`and preferably means that codons encoding positively-charged residues (H, K, and R) are
`
`substituted with codons encoding positively-charged residues, codons encoding negatively-
`charged residues (D and E) are substituted with codons encoding negatively-charged residues,
`codons encoding neutral polar residues (C, G, N, Q, S, T, and Y) are substituted with codons
`
`encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L,
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`11
`
`M, P, V, and W) are substituted with codons encoding neutral non-polar residues. These
`
`variations can spontaneously occur, be introduced by random mutagenesis, or can be
`introduced by directed mutagenesis. Those changes can be made without destroying the
`essential characteristics of these polypeptides, which are to recognize antitumor antigens in
`the context of an MHC with high avidity so as to enable the destruction of cancer cells. The
`
`ordinarily skilled artisan can readily and routinely screen variant amino acids and/or the
`nucleic acids encoding them to determineif these variations substantially lessen or destroy the
`ligand binding capacity by methods knownin theart.
`
`As outlined above, the TCR nucleic sequences may have been altered in order to provide
`codon optimization. Codon optimization is a generic technique to achieve optimal expression
`of a foreign gene in a cell system. Selection of optimum codons depends on codon usage of
`the host genome and the presence of several desirable and undesirable sequence motifs. It is
`noted that codon optimization will not lead to an altered amino acid sequence and,thus, will
`notfall under the definition of a conservative substitution as contained in this application.
`
`In a still further embodiment, the vectors contain nucleic acids coding for functional TCR a
`
`and/or B chain fusion proteins, comprising:
`
`a) at least one epitope-tag, and
`
`b) the amino acid sequence of an o and/or 8 chain of a TCR as defined hereinabove,
`
`wherein said epitope-tag is selected from
`
`i) an epitope-tag added to the N- and/or C-terminus of said @ and/or B chain, or added into the
`a and/or B chain sequence, but outside the CDR3region,
`
`ii) an epitope-tag inserted into a constant region of said a and/or B chain, and
`
`iii) an epitope-tag replacing a number of amino acids in a constant region of said a and/or B
`chain.
`
`Epitope tags are short stretches of amino acids to which a specific antibody can be raised,
`which in some embodiments allows one to specifically identify and track the tagged protein
`that has been addedto a living organism orto cultured cells. Detection of the tagged molecule
`can be achieved using a numberof different techniques. Examples of such techniques include:
`immunohistochemistry,
`immunoprecipitation, flow cytometry,
`immunofluorescence micro-
`
`scopy, ELISA, immunoblotting ("Western"), and affinity chromatography. Epitope tags add a
`known epitope (antibody binding site) on the subject protein, to provide binding of a known
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`12
`
`and often high-affinity antibody, and thereby allowing one to specifically identify and track
`the tagged protein that has been addedto a living organism orto cultured cells.
`
`In the context of the present invention, a “functional” T-cell receptor (TCR) o- and/or B-chain
`fusion protein shall mean an a- and/or B-chain fusion protein that, although the chain includes
`the epitope-tag and/or has a tag attached to it, maintains at least substantial fusion protein
`biological activity in the fusion. In the case of the a- and/or B-chain of a TCR,this shall mean
`that both chains remain able to form a T-cell receptor (either with a non-modified a- and/or B-
`chain or with another inventive fusion protein a- and/or B-chain) which exerts its biological
`function, in particular binding to the specific peptide-MHC complex of said TCR, and/or
`functional signal transduction upon peptide activation.
`
`Preferred is a functional T-cell receptor (TCR) a- and/or B-chain fusion protein according to
`the present invention, wherein said epitope-tag has a length of between 6 to 15 aminoacids,
`preferably 9 to 11 amino acids.
`
`Even more preferred is a functional T-cell receptor (TCR) a- and/or B-chain fusion protein
`according to the present invention, wherein said T-cell receptor (TCR) a- and/or B-chain
`fusion protein comprises two or more epitope-tags, either spaced apart or directly in tandem.
`Embodiments of the fusion protein can contain 2, 3, 4, 5 or even more epitope-tags, as long as
`the fusion protein maintainsits biological activity/activities (“functional”).
`
`Preferred is a functional T-cell receptor (TCR) o- and/or B-chain fusion protein according to
`the present invention, wherein said epitope-tag is selected from, but not limited to, CD20 or
`Her2/neu tags, or other conventional
`tags such as a myc-tag, FLAG-tag, T7-tag, HA
`(hemagglutinin)-tag, His-tag, S-tag, GST-tag, or GFP-tag. The myc, T7, GST, GFP tags are
`epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag
`designed for high antigenicity (see, e.g., U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc
`tag can preferably be used because high quality reagents are available to be used forits
`detection. Epitope tags can of course have one or more additional
`functions, beyond
`recognition by an antibody. The sequences of these tags are described in the literature and
`well known to the person of skill in art.
`
`In the functional T-cell receptor (TCR) a- and/or B-chain fusion protein according to the
`
`

`

`WO 2010/089412
`
`PCT/EP2010/051565
`
`13
`
`invention, said fusion protem may be for example selected from two myc-tag
`present
`sequences that are attached to the N-terminus of an a-TCR-chain and/or 10 amino acids of a
`
`protruding loop region in the f-chain constant domain being exchanged for the sequence of
`
`two myc-tags.
`
`In an embodimentof the present invention, the inventors inserted an amino acid sequencethat
`
`correspondsto a part of the myc protein (myc-tag) at several reasonable sites into the structure
`
`of a T cell receptor and transduced this modified receptor into T cells (see examples below).
`
`By introducing a tag into the TCR structure, it is possible to depicte the modified cells by
`administering the tag-specific antibodyto the patient.
`
`Those functional TCR fusion proteins may be used in a method for selecting a host cell
`population expressing a fusion protein selected from the group consisting of a fusion protein
`comprising a) at least one epitope-providing amino acid sequence (epitope-tag), and b) the
`amino acid sequence of an a- and/or B-chain of a TCR as defined above, wherein said epitope-
`tag is selected from an epitope-tag added to the N- and/or C-terminus of said a- and/or B-
`chain or added into the a- and/or B-chain sequence, but outside the CDR3 region, an epitope-
`tag inserted into a constant region of said a- and/or B-chain, and an epitope-tag replacing a
`number of amino acids in a constant region of said a- and/or B-chain; and a TCR comprising
`at least one fusion protein as above on the surface of thehostcell; comprising contacting host
`cells in a