DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 166-185 are pending and under examination in the instant office action.
Information Disclosure Statement
Applicants are kindly reminded of their duty to disclose pursuant to 37 C.F.R. 1.56 which encompasses the citation of references material to patentability of which Applicants are aware, such as references that may have been cited in the International Search Report of the parent applicant or in the specification.
Specification
The disclosure is objected to because of the following informalities:
The instant specification at least at paragraphs [0009], [0017], [0035], [0110], [0141] teaches that an NES comprises a "polynucleotide pattern" and then contradicts the teachings of a "polynucleotide pattern" by disclosing particular amino acids and polypeptide sequences.
The instant specification re-defines the amino acid abbreviation “L” as “a hydrophobic amino acid residue” and “the hydrophobic amino acid residue is selected from the group consisting of leucine, isoleucine, valine, phenylalanine, and methionine” at least at paragraphs [0009], [0017], [0035], [0110], [0141]. However, the specification also teaches particular sequences SEQ ID NO: 2 and 3, wherein “L” is defined as leucine (see sequence listing and Standards ST.26, Annex I ‘Controlled Vocabulary’). “L” is art-understood to designate the particular amino acid leucine; it is repugnant in the art to uses "L" to mean anything other than leucine when describing an amino acid residue. The wildcard to define “a hydrophobic amino acid” should be designated as another letter or symbol not art-understood as a single amino acid.
The specification at Example 8 and Example 9 teaches, for example, “In some examples, the systems 110 and 130 as illustrated in FIG. 1A may be expressed in immune cells (e.g. T cells), and the engineered immune cells may be tested in vivo in a mouse tumor model to study their anti-tumor efficacy. The system 110 may be referred to as “Conv CAR” in this Example” ([0330], also see similar language in [0342]). Later, the Examples teach “conventional HER2-CAR-transduced T cells” [0332]. This is objected to because it is not possible to discern in the Example and Figures whether wildtype LAT is overexpressed (exogenous LAT) as in one embodiment of system 110, or if the cells labeled “Conv CAR” are only transduced with HER2-CAR and rely on endogenous levels of LAT.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 170-174, 181-182, and 184 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 170-174 and 181-182 are rejected for recitation of intended result/effect without conferring some structural, material, or manipulative difference on the scope of the claim. Claim 170 recites “wherein upon introduction of the chimeric polypeptide into a cell comprising the receptor, the chimeric polypeptide prolongs or enhances signaling of the receptor in the cell, as compared to the adaptor protein without the plurality of heterologous NES domains”; claim 171 recites “wherein the plurality of heterologous NES domains enhances translocation of the adaptor protein into a membrane of the cell”; claim 172 recites “wherein the plurality of heterologous NES domains reduces displacement of the adaptor protein from a membrane of the cell during the signaling of the receptor”; claim 173 recites “wherein the plurality of heterologous NES domains reduces degradation of the adaptor protein during the signaling of the receptor”; and claim 174 recites “wherein the plurality of heterologous NES domains stabilizes the adaptor protein in a membrane of the cell, as compared to the adaptor protein without the plurality of heterologous NES domains”. Apart from the structure of the chimeric protein comprising adaptor protein comprising at least a portion of LAT and a plurality of heterologous NESs, there is no further structure described in the claim or the specification that would apprise a person of skill in the art which structures are encompassed by the claim. Claims 181 and 182 are directed towards the chimeric polypeptide of claim 166 comprising one additional polypeptide, “wherein upon introduction of the chimeric polypeptide into a cell, a charge, size, and/or position of the at least one additional polypeptide relative to the chimeric polypeptide is sufficient to inhibit or reduce the interaction between the adaptor protein and a cellular component of the cell”. Absent specific structures in the claims or specification that are clearly linked to these functions, it is unclear how these claims further limit the scope of the parent claim. MPEP 2173.05(g) states: “the use of functional language in a claim may fail ‘to provide a clear-cut indication of the scope of the subject matter embraced by the claim' and thus be indefinite.” It further states: “Examiners should consider the following factors when examining claims that contain functional language to determine whether the language is ambiguous: (1) whether there is a clear cut indication of the scope of the subject matter covered by the claim; (2) whether the language sets forth well-defined boundaries of the invention or only states a problem solved or a result obtained; and (3) whether one of ordinary skill in the art would know from the claim terms what structure or steps are encompassed by the claim” (emphasis added). Since the claims fail to meet all (3) criteria set forth in MPEP 2173.05(g), then claims 170-174 and 181-182 are rejected.
The term “enhances” in claim 171 is a relative term which renders the claim indefinite. The term “enhances” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which translocation of the adaptor protein into the membrane of the cell would be enhanced in order to meet the limitations of the claim.
The term “enhances” and “prolongs” and “enhances” in claims 170-171, respectively, is a relative term which renders the claim indefinite. The term “enhances” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which signaling of the receptor or translocation of the adaptor protein into the membrane of the cell would be enhanced in order to meet the limitations of the claim.
The term “enhances” and “prolongs” and “enhances” in claims 170-171 respectively is a relative term which renders the claim indefinite. The term “enhances” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which signaling of the receptor or translocation of the adaptor protein into the membrane of the cell would be enhanced in order to meet the limitations of the claim.
The term “reduces” in claims 172-173 respectively is a relative term which renders the claim indefinite. The term “reduces” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which displacement of the adaptor protein from a membrane or degradation of the adaptor protein during signaling must be reduced in order to meet the limitations of the claims.
The term “stabilizes” in claims 174 is a relative term which renders the claim indefinite. The term “stabilizes” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which stabilization of the adaptor protein in the membrane as compared to the adaptor protein as compared to the adaptor protein without the plurality of heterologous NES domains must occur to meet the limitations of the claim.
Claims 170, 174, and 182 are indefinite for the recitation of “as compared to the adaptor protein without the plurality of heterologous NES domains” and “as compared to the adaptor protein without the at least one additional polypeptide”. This is unclear because claim 166 from which claims 170 and 174 depends recites “a chimeric polypeptide comprising a plurality of heterologous nuclear export signal (NES) domains linked to an adaptor protein of a receptor”, but it does not recite an adaptor protein comprising a plurality of heterologous NES domains; claim 181 from which claim 182 depends recites that the chimeric polypeptide comprises at least one additional polypeptide, but does not recite an adaptor protein comprising an additional polypeptide. Therefore, it is unclear whether the claim requires a control of the chimeric polypeptide without the NES domains or at least one additional polypeptide, just the adaptor protein without the NES domains or at least one additional polypeptide, or something else.
Claims 175-177 are indefinite for the recitation of a heterologous NES domain comprising a polynucleotide sequence. Nuclear export signal (NES) domains are understood to be a polypeptide domain, and further the instant claims recite the polynucleotide comprising particular amino acids. Therefore, the metes and bounds of the claim are unclear.
Claims 175-177 are indefinite for the recitation of “having the pattern […] wherein each L is a hydrophobic amino acid residue selected from the group consisting of leucine, isoleucine, valine, phenylalanine, and methionine”. However, the specification also teaches particular sequences SEQ ID NO: 2 and 3, wherein “L” is defined as leucine (see sequence listing and Standards ST.26, Annex I ‘Controlled Vocabulary’). “L” is art-understood to designate the particular amino acid leucine; it is repugnant in the art to uses "L" to mean anything other than leucine when describing an amino acid residue. The wildcard to define “a hydrophobic amino acid” should be designated as another letter or symbol not art-understood as a single amino acid.
Claims 175-177 are indefinite for the recitation of having the patterns “LxxLxL”, “LxxxLxL”, and “LxxxLxxLxL”, respectively. While the claims define “L”, there is no definition in the claim or specification for what is represented by the lowercase x. Therefore, the metes and bounds of the claim are unclear.
The term “reduce[s]” in claims 181 and 182 and “inhibit” in claim 181 is a relative term which renders the claim indefinite. The terms “reduce[s]” and “inhibit” are not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear the degree to which reduction or inhibition of the interaction between the adaptor protein and a cellular component of the cell or reduction of degradation of the adaptor protein in the cell must occur in order to meet the limitations of the claims.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 166-185 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Regarding claim 166, the claim is directed towards: 1) “a plurality of heterologous nuclear export signal domains”, but the instant specification fails to sufficiently describe a genus of chimeric polypeptides proteins comprising a plurality of any NES domains at any position within the chimeric polypeptide and 2) “wherein the adaptor protein comprises at least a portion of a Linker for Activation of T cells (LAT)” (emphasis is the examiner’s).
Regarding claim 173, the claim is directed towards the chimeric polypeptide wherein the upon the introduction of the chimeric polypeptide into a cell comprising the receptor, the polypeptide prolongs or enhances signaling and the NES domains reduce degradation of the adaptor protein during the signaling of the receptor. As described in the 112(b) above, this is indefinite for the recitation of intended result without a clear link to structure. In addition, the specification does not sufficiently describe the genus, or any species at all, that are clearly shown to reduce the degradation of the adaptor protein.
Regarding claim 181, the claim is directed towards the polypeptide of claim 166 wherein the polypeptide further comprises at least one additional polypeptide, wherein upon introduction of the chimeric polypeptide into any cell, any charge, size, and/or position of the at least one additional polypeptide relative to the chimeric polypeptide is sufficient to inhibit of reduce interaction between the adaptor protein and any cellular component of the cell, where there is not description of the interaction between the one additional polypeptide of the chimeric polypeptide and any cellular component.
Regarding claim 183, the claim is directed towards the chimeric polypeptide comprising any recognition moiety that is specifically recognized by any antibody; nearly any polypeptide sequence may be specifically recognized by an antibody. There is no further definition of recognition moiety in the specification.
Scope of the claimed genus
Claim 166 is directed towards: 1) “a plurality of heterologous nuclear export signal domains”, wherein the NES domains may be any NES domain and may be positioned anywhere within the chimeric protein and an adaptor protein of any receptor 2) “wherein the adaptor protein comprises at least a portion of a Linker for Activation of T cells (LAT)” (emphasis is the examiner’s). The instant specification does not define “portion”, and therefore the invention may encompass any portion of LAT, even only a few amino acids.
Regarding claims 175-177, the claims are directed towards a NES comprising the patterns “LxxLxL”, “LxxxLxL”, and “LxxxLxxLxL”, respectively. These are indefinite as described in the 112(b) rejection above, but for the purposes of expedited prosecution, these patterns will be considered to be polypeptide patterns wherein “L” is a hydrophobic amino acid selected from the group of leucine, isoleucine, valine, phenylalanine, and methionine as defined by the claims, and “x” will be considered to be any single amino acid. However, these claims do not further limit the portion of LAT or the position of the NES relative to the LAT.
Regarding claims 178-180, the claims recite a chimeric polypeptide wherein the portion of the LAT comprises at least one mutation as compared to a wild-type LAT; wherein the at least one mutation is at one or more cysteine residues of the wild-type LAT; and wherein the mutation is at one or more lysine residues of the wild-type LAT.
Regarding claims 181, the claim is directed towards the polypeptide of claim 166 wherein the polypeptide further comprises at least one additional polypeptide, wherein upon introduction of the chimeric polypeptide into any cell, any charge, size, and/or position of the at least one additional polypeptide relative to the chimeric polypeptide is sufficient to inhibit of reduce interaction between the adaptor protein and any cellular component of the cell. Regarding claim 182, the claim recites several limitations in the alternate including several functional limitations of the one additional polypeptide, or limits the cellular component to any nucleic acid, any polynucleotide, any amino acid, any polypeptide, any lipid, any carbohydrate, any small molecule, any enzyme, any ribosome, any proteasome, any variant thereof, or any combination thereof.
Regarding claims 167, 170-174, and 181-185, the claims depend on claim 166 without further limiting the structure of the portion of LAT of the adaptor protein or the NES.
State of the Relevant Art
Chimeric polypeptides comprising adaptor proteins and some of the functions of the LAT protein are known in the art. LAT is a known adaptor protein that is recruited to coordinate the intracellular signaling that is triggered upon T cell activation when phosphorylated Zap-70 in turn phosphorylates LAT a conserved tyrosine residues (Simeoni, Luca, et al. "Control of lymphocyte development and activation by negative regulatory transmembrane adapter proteins." Immunological reviews 224.1 (2008): 215-228, See “TRAPs that regulate T-cell development: LAT” section p. 216-218). Lo, WL., Shah, N.H., Ahsan, N. et al. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol 19, 733–741 (2018). https://doi.org/10.1038/s41590-018-0131-1 teaches that particular domains of LAT, a membrane-proximal proline-rich motif, are critical for LAT phosphorylation and TCR signaling. The negatively charged portion of LAT between amino acids 113 and 126 has been shown to be required for LAT interaction with Lck, but causes an increase in phosphorylation of LAT and ZAP70 phosphorylation (Arbulo-Echevarria, Mikel M., et al. "A stretch of negatively charged amino acids of linker for activation of t-cell adaptor has a dual role in t-cell antigen receptor intracellular signaling." Frontiers in immunology 9 (2018): 115). In addition, Torgersen, Knut Martin, et al. "A soluble LAT deletion mutant inhibits T-cell activation: reduced recruitment of signalling molecules to glycolipid-enriched microdomains." Cellular Signalling 13.3 (2001): 213-220 teaches that a mutant of LAT wherein the transmembrane domain is deleted inhibits TCR signaling by reducing interaction of endogenous LAT with PLC-y1 and Grb2 (Abstract). This suggests that although roles are known for different portions of LAT, it was still unclear at the time of filing that any individual domain of LAT was necessary and sufficient for a particular signaling role in the absence of the rest of the molecule. Despite some structure/function characterization of LAT known in the art, LAT was not completely characterized. For example, post-filing publication James, et al. "Redox regulation of LAT enhances T cell-mediated inflammation." Antioxidants 13.4 (2024): 499 teaches that the effects of redox on LAT are not well studied and “redox insensitivity of LAT leads to a marked reduction in CD4+ CD8+ T cells in the thymus, reminiscent of the phenotypes observed in thymocytes deficient in other LAT signalosome molecules such as LAT, LCK, or SLP-76” (Discussion ¶4). This redox-insensitive LAT was created by mutation of two particular cysteines C120 and C172 because cysteines C26 and C29 had been previously shown to be required for proper incorporation into the plasma membrane (Discussion ¶2). Pham-Danis, Catherine, et al. "Restoration of LAT activity improves CAR T cell sensitivity and persistence in response to antigen-low acute lymphoblastic leukemia." Cancer Cell 43.3 (2025): 482-502 teaches that coupling of the intracellular signaling domain of LAT to a traditional CAR extracellular domain increases the efficiency of LAT activation increase the LAT activity of a second-generation CAR and increased efficacy and persistence in response to CD22-low B-ALL (Abstract). There is no art of record that describes a minimal portion of LAT smaller than the entire LAT protein for targeting of LAT to an active CAR or TCR, or for regulation of TCR signaling.
Regarding regulation of LAT activity via inhibiting proteasome-mediated degradation of LAT, ubiquitylation-resistant mutants of LAT by substitution of K52 and K204 in human LAT with arginine resulted in mutants that were defective in LAT turnover and displayed elevated T cell signaling (L. Balagopalan, et. al., Enhanced T-cell signaling in cells bearing linker for activation of T-cell (LAT) molecules resistant to ubiquitylation, Proc. Natl. Acad. Sci. U.S.A. 108 (7) 2885-2890, https://doi.org/10.1073/pnas.1007098108 (2011), See p. 2885 right column, Fig. 3, Fig. 4). There is no art of record that describes steric or charge-based inhibition of LAT degradation.
Regarding nuclear export sequences, there is no art of record regarding nuclear export sequences that inhibit proteasome degradation. In fact, although there is no art in particular teaching NES or polypeptides comprising leucine are related to degradation, it has been shown that exposed hydrophobic patches can destabilize a protein and increase degradation (Tae HS, et. al., Identification of hydrophobic tags for the degradation of stabilized proteins. Chembiochem. 2012 Mar 5;13(4):538-41. doi: 10.1002/cbic.201100793. Epub 2012 Jan 23. PMID: 22271667; PMCID: PMC3338156). Thus, it appears that any polypeptide fitting into the patterns “LxxLxL”, “LxxxLxL”, or “LxxxLxxLxL”, which includes embodiments of long stretches of hydrophobic amino acid, may also destabilize the instant chimeric adaptor rather than perform the intended function except for those specifically described in the application. Regarding the predictability of NES signals, false positives are common with a consensus sequence including all hydrophobic residues (La Cour, Tanja, et al. "Analysis and prediction of leucine-rich nuclear export signals." Protein Engineering Design and Selection 17.6 (2004): 527-536, see p. 529 right column). Therefore, although there is an apparent consensus sequence, typically an effective nuclear export signal must be experimentally determined. Typically, a nuclear export sequence must interact with the nuclear export protein exportin-1 (CRM1) which is located in the cytoplasm and nucleoplasm to participate in shuttling through the nuclear pore (La Cour et. al., p. 527 “Introduction”). Therefore, in relation to the function of the instant adaptor protein comprising at least a portion of LAT, a structure/function relationship is not disclosed in the art that would describe any NES sequence in any position of the chimeric adaptor protein.
Summary of Species disclosed in the original specification
The instant specification teaches working examples of chimeric adaptor proteins comprising full-length human LAT of the configuration N-NES1-LAT-NES2-(cleavable linker)-dCas9-KRAB as shown in 130, Fig. 1A. This system improves CD4+ T cell proliferation when when co-expressed with HER2CAR-TEV (a protease-linked CAR that cleaves the linker to release Cas9) as compared to a conventional HER2 CAR expressed alone or a HER2CAR TEV expressed alone (Fig. 1B) and increases cytotoxic activity against tumor cells (Fig. 2, Fig. 3). This system is further tested in an in vivo tumor model with and without PD-1 guide RNA and compared to a conventional HER2 CAR, where increased mouse lifespan (Fig. 12E, Fig. 13B) and reduced tumor size compared to the conventional CAR alone (Fig. 12C). The specification also prophetically discloses embodiments of the chimeric adapter protein that do not include the additional dCas9-KRAB, but none of these embodiments are made or tested (Fig. 10B, Fig. 14). The specification also prophetically discloses versions wherein LAT is mutated at residues C9, C26, C29, and/or C117 to make ‘redox insensitive’ LAT (Fig. 9C) and prophetically discloses mutation of lysine residues to prevent ubiquitination and degradation of the adaptor protein [0144].
There are no embodiments wherein the NESs are only C-terminal or only N-terminal, or are disposed between domains of the adaptor protein. There are no embodiments comprising less than full-length LAT. Additionally, no embodiments are tested wherein the accompanying CAR is not linked to the TEV protease and therefore is expressed at a higher level, which would be expected to affect T-cell activation.
Identifying characteristics and structure/function correlation
MPEP § 2163 states that a “representative number of species” means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. As applicants have only described one species of the chimeric adaptor protein, which comprising one extracellular NES, full-length LAT, one intracellular NES, a TEV cleavage site linker, and dCAS9-KRAB, it is not possible to discern from the instant application what species of the instantly claimed invention would possess the functional properties described and claimed. For instance, applicants prophetically describe an N-terminal NES that decreases ubiquitination and proteasomal degradation of LAT Fig. 6(iii). However, there is no evidence in the art or the instant specification that would support whether an extracellular NES alone would perform the instantly claimed function. It is not discernable from the art or the instant specification which portion of LAT, which position or number of NESs are required for the instant function (excepting the instantly tested species), or even whether the NES alone is responsible for the claimed decrease in degradation rather than the steric hinderance of the dCas9KRAB of the tested variants is responsible (Fig. 7). Indeed, the instant specification does not even measure the levels of the instant chimeric adaptor protein; the only data that is shown is that the non-claimed chimeric antigen receptor that works in concert with the chimeric adaptor protein of the instant species is co-expressed (Fig. 4B). There is no evidence of any particular domain or portion of the protein that could describe a structure/function relationship for this phenomenon.
Summary
A genus of species is not present in the instant specification or prior art that would demonstrate a structure/activity relationship would be known the instant chimeric adaptor protein to determine which species of the genus possess the instant utility of increasing CAR or TCR signaling. There is a lack of an appropriate number of species with altered NES signal sequences, domain positions, truncations or portions of LAT, mutations in the LAT domain, or lacking a C-terminal dCAS9-KRAB domain. One of skill in the art would reasonably conclude that the applicant was not in possession of the genus of different chimeric adaptor proteins of claim 166 at the time of filing. There is a lack of “additional portions” in different positions with different charges or sizes to describe the function of disrupting any interaction with any cellular component. Regarding claims 167-185 the claims are ultimately dependent on the rejected claim 166 without narrowing the claimed subject matter and thus are also rejected.
Claim Rejections - 35 USC § 112(a)- Scope of Enablement
Claims 166-185 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
a chimeric polypeptide comprising a plurality of heterologous nuclear export signal (NES) domains linked to an adaptor protein of a receptor, wherein the adaptor protein comprises full-length Linker for Activation of T cells (LAT);
wherein the plurality of heterologous NES domains comprises at least a first and a second NES domain and wherein the adaptor protein is disposed between the first heterologous NES domain and the second heterologous NES domain;
and wherein the chimeric polypeptide further comprises an additional C-terminal polypeptide comprising a gene modulating polypeptide (GMP) comprising dCas9-KRAB,
does not reasonably provide enablement for:
a chimeric polypeptide comprising any plurality of heterologous nuclear export signal (NES) domains linked to an adaptor protein of a receptor in any way and at any position; wherein the adaptor protein comprises any portion of Linker for Activation of T cells (LAT), and wherein the chimeric polypeptide may or may not comprise any additional components.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is "undue." These
factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Scope of the claimed genus and nature of the invention
Claim 166 is directed towards: 1) “a plurality of heterologous nuclear export signal domains”, wherein the NES domains may be any NES domain and may be positioned anywhere within the chimeric protein and an adaptor protein of any receptor 2) “wherein the adaptor protein comprises at least a portion of a Linker for Activation of T cells (LAT)” (emphasis is the examiner’s). The instant specification does not define “portion”, and therefore the invention may encompass any portion of LAT, even only a few amino acids.
Regarding claims 170-174, the claims are directed towards intended functional outcomes of the chimeric polypeptide “wherein upon introduction of the chimeric polypeptide into a cell comprising the receptor, the chimeric polypeptide prolongs or enhances signaling of the receptor in the cell as compared to the adaptor protein without the plurality of heterologous NES domains” (see also the 112(b) rejection above). Claim 173 also recites wherein any plurality of heterologous NES domains reduces degradation of the adaptor protein during the signaling of the receptor.
Regarding claims 175-177, the claims are directed towards a NES comprising the patterns “LxxLxL”, “LxxxLxL”, and “LxxxLxxLxL”, respectively. These are indefinite as described in the 112(b) rejection above, but for the purposes of expedited prosecution, these patterns will be considered to be polypeptide patterns wherein “L” is a hydrophobic amino acid selected from the group of leucine, isoleucine, valine, phenylalanine, and methionine as defined by the claims, and “x” will be considered to be any single amino acid. However, these claims do not further limit the portion of LAT or the position of the NES relative to the LAT.
Regarding claims 178-180, the claims recite a chimeric polypeptide wherein the portion of the LAT comprises at least one mutation as compared to a wild-type LAT; wherein the at least one mutation is at one or more cysteine residues of the wild-type LAT; and wherein the mutation is at one or more lysine residues of the wild-type LAT.
Regarding claims 181, the claim is directed towards the polypeptide of claim 166 wherein the polypeptide further comprises at least one additional polypeptide, wherein upon introduction of the chimeric polypeptide into any cell, any charge, size, and/or position of the at least one additional polypeptide relative to the chimeric polypeptide is sufficient to inhibit of reduce interaction between the adaptor protein and any cellular component of the cell. Regarding claim 182, the claim recites several limitations in the alternate including several functional limitations of the one additional polypeptide, or limits the cellular component to any nucleic acid, any polynucleotide, any amino acid, any polypeptide, any lipid, any carbohydrate, any small molecule, any enzyme, any ribosome, any proteasome, any variant thereof, or any combination thereof.
Regarding claims 167, 170-174, and 181-185, the claims depend on claim 166 without further limiting the structure of the portion of LAT of the adaptor protein or the NES.
State of the Relevant Art; level of one of ordinary skill; and level of predictability of the art
As described in the written description rejection above, chimeric polypeptides comprising adaptor proteins and some of the functions of the LAT protein are known in the art. LAT is a known adaptor protein that is recruited to coordinate the intracellular signaling that is triggered upon T cell activation when phosphorylated Zap-70 in turn phosphorylates LAT a conserved tyrosine residues (Simeoni, Luca, et al. "Control of lymphocyte development and activation by negative regulatory transmembrane adapter proteins." Immunological reviews 224.1 (2008): 215-228, See “TRAPs that regulate T-cell development: LAT” section p. 216-218). Lo, WL., Shah, N.H., Ahsan, N. et al. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol 19, 733–741 (2018). https://doi.org/10.1038/s41590-018-0131-1 teaches that particular domains of LAT, a membrane-proximal proline-rich motif, are critical for LAT phosphorylation and TCR signaling. The negatively charged portion of LAT between amino acids 113 and 126 has been shown to be required for LAT interaction with Lck, but causes an increase in phosphorylation of LAT and ZAP70 phosphorylation (Arbulo-Echevarria, Mikel M., et al. "A stretch of negatively charged amino acids of linker for activation of t-cell adaptor has a dual role in t-cell antigen receptor intracellular signaling." Frontiers in immunology 9 (2018): 115). In addition, Torgersen, Knut Martin, et al. "A soluble LAT deletion mutant inhibits T-cell activation: reduced recruitment of signalling molecules to glycolipid-enriched microdomains." Cellular Signalling 13.3 (2001): 213-220 teaches that a mutant of LAT wherein the transmembrane domain is deleted inhibits TCR signaling by reducing interaction of endogenous LAT with PLC-y1 and Grb2 (Abstract). This suggests that although roles are known for different portions of LAT, it was still unclear at the time of filing that any individual domain of LAT was necessary and sufficient for a particular signaling role in the absence of the rest of the molecule. Despite some structure/function characterization of LAT known in the art, LAT was not completely characterized. For example, post-filing publication James, Jaime, et al. "Redox regulation of LAT enhances T cell-mediated inflammation." Antioxidants 13.4 (2024): 499 teaches that the effects of redox on LAT are not well studied and “redox insensitivity of LAT leads to a marked reduction in CD4+ CD8+ T cells in the thymus, reminiscent of the phenotypes observed in thymocytes deficient in other LAT signalosome molecules such as LAT, LCK, or SLP-76” (Discussion ¶4). This redox-insensitive LAT was created by mutation of two particular cysteines C120 and C172 because cysteines C26 and C29 had been previously shown to be required for proper incorporation into the plasma membrane (Discussion ¶2). Pham-Danis, Catherine, et al. "Restoration of LAT activity improves CAR T cell sensitivity and persistence in response to antigen-low acute lymphoblastic leukemia." Cancer Cell 43.3 (2025): 482-502 teaches that coupling of the intracellular signaling domain of LAT to a traditional CAR extracellular domain increases the efficiency of LAT activation increase the LAT activity of a second-generation CAR and increased efficacy and persistence in response to CD22-low B-ALL (Abstract). There is no art of record that describes a minimal portion of LAT smaller than the entire LAT protein for targeting of LAT to an active CAR or TCR, or for regulation of TCR signaling.
Regarding regulation of LAT activity via inhibiting proteasome-mediated degradation of LAT, ubiquitylation-resistant mutants of LAT by substitution of K52 and K204 in human LAT with arginine resulted in mutants that were defective in LAT turnover and displayed elevated T cell signaling (L. Balagopalan, et. al., Enhanced T-cell signaling in cells bearing linker for activation of T-cell (LAT) molecules resistant to ubiquitylation, Proc. Natl. Acad. Sci. U.S.A. 108 (7) 2885-2890, https://doi.org/10.1073/pnas.1007098108 (2011), See p. 2885 right column, Fig. 3, Fig. 4). There is no art of record that describes steric or charge-based inhibition of LAT degradation.
Regarding nuclear export sequences, there is no art of record regarding nuclear export sequences that inhibit proteasome degradation. In fact, although there is no art in particular teaching NES or polypeptides comprising leucine are related to degradation, it has been shown that exposed hydrophobic patches can destabilize a protein and increase degradation (Tae HS, et. al., Identification of hydrophobic tags for the degradation of stabilized proteins. Chembiochem. 2012 Mar 5;13(4):538-41. doi: 10.1002/cbic.201100793. Epub 2012 Jan 23. PMID: 22271667; PMCID: PMC3338156). Thus, it appears that any polypeptide fitting into the patterns “LxxLxL”, “LxxxLxL”, or “LxxxLxxLxL”, which includes embodiments of long stretches of hydrophobic amino acid, may also destabilize the instant chimeric adaptor rather than perform the intended function except for those specifically described in the application. Regarding the predictability of NES signals, false positives are common with a consensus sequence including all hydrophobic residues (La Cour, Tanja, et al. "Analysis and prediction of leucine-rich nuclear export signals." Protein Engineering Design and Selection 17.6 (2004): 527-536, see p. 529 right column). Therefore, although there is an apparent consensus sequence, typically an effective nuclear export signal must be experimentally determined. Typically, a nuclear export sequence must interact with the nuclear export protein exportin-1 (CRM1) which is located in the cytoplasm and nucleoplasm to participate in shuttling through the nuclear pore (La Cour et. al., p. 527 “Introduction”). Therefore, in relation to the function of the instant adaptor protein comprising at least a portion of LAT, it would not have been predictable which NESs or which positions would increase LAT functions as described, or would increase the CAR-TEV expression as shown; or which portions of LAT are required for the increase in T cell function.
Summary of Species disclosed in the original specification; the amount of direction provided by the inventor, existence of working examples; and quality of experimentation needed to make or use the invention based on the content of the disclosure.
The instant specification teaches working examples of chimeric adaptor proteins comprising full-length human LAT of the configuration N-NES1-LAT-NES2-(cleavable linker)-dCas9-KRAB as shown in 130, Fig. 1A. This system improves CD4+ T cell proliferation when when co-expressed with HER2CAR-TEV (a protease-linked CAR that cleaves the linker to release Cas9) as compared to a conventional HER2 CAR expressed alone or a HER2CAR TEV expressed alone (Fig. 1B) and increases cytotoxic activity against tumor cells (Fig. 2, Fig. 3). This system is further tested in an in vivo tumor model with and without PD-1 guide RNA and compared to a conventional HER2 CAR, where increased mouse lifespan (Fig. 12E, Fig. 13B) and reduced tumor size compared to the conventional CAR alone (Fig. 12C). The specification also prophetically discloses embodiments of the chimeric adapter protein that do not include the additional dCas9-KRAB, but none of these embodiments are made or tested (Fig. 10B, Fig. 14). The specification also prophetically discloses versions wherein LAT is mutated at residues C9, C26, C29, and/or C117 to make ‘redox insensitive’ LAT (Fig. 9C) and prophetically discloses mutation of lysine residues to prevent ubiquitination and degradation of the adaptor protein [0144]. However, even for the tested embodiment, there is no data to suggest which or if any of the domains of the chimeric polypeptide are required for the prophetically recited mechanisms and functions. Applicant suggests that there decrease in LAT displacement from the membrane and subsequent proteasomal degradation, but there are no measurement of the levels of chimeric adaptor polypeptide expression levels for any embodiment of the invention. There are no controls where LAT is overexpressed separate from the specific embodiment tested. As such, it is impossible to determine from the instant specification which embodiments of the claimed invention, apart from N-NES1-LAT-NES2-(cleavable linker)-dCas9-KRAB, would work to enhance T cell function. The inventors prophetically recite that the NES blocks proteasomal degradation of a chimeric adaptor protein comprising any portion of LAT, but there is no evidence that there is more of the instant receptor on the plasma membrane other than indirect measurements of increased CAR expression and increased T cell signaling. How is a person of skill in the art expected to determine whether or not the c-terminal dCas9-KRAB is required for the increase T cell signaling shown? Applicant suggests both the NES and dCas9-KRAB could block proteasomal degradation of LAT, but there is no evidence for one or the other, or both are required. Further, it is not clear what the minimal domain(s) for a portion of LAT would be required to perform the instant T cell signaling functions.
There are no embodiments wherein the NESs are only C-terminal or only N-terminal, or are disposed between domains of the adaptor protein. There are no embodiments comprising less than full-length LAT. There are no embodiments, other than prophetically recited, where the chimeric adaptor protein lacks the dCas9-KRAB. Additionally, no embodiments are tested wherein the accompanying CAR is not linked to the TEV protease and therefore is expressed at a higher level, which would be expected to affect T-cell activation.
As such, every single embodiment in the genus of that is not the one shown in part 130 of Fig. 1A would have to be independently tested in order to determine which embodiments of the invention are effective.
Conclusion
The applicant does not have enablement for the full scope of the chimeric adaptor protein as claimed in claims 166-185. It would take undue experimentation in order to determine which embodiments of the chimeric adaptor protein would perform the functions described in the specification.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 166-168, 175, 177, and 185 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by U.S. 20190203227 to Ho et. al as evidenced by Uniprot O43561 LAT_Human and Lo, WL., Shah, N.H., Ahsan, N. et al. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol 19, 733–741 (2018). https://doi.org/10.1038/s41590-018-0131-1.
Instant claim 166 is directed towards a chimeric polypeptide comprising a plurality of heterologous nuclear export domains linked to an adaptor protein of a receptor, wherein the adaptor protein comprises at least a portion of a Linker for Activation of T cells. The instant specification does not further restrict the definition of an adaptor protein of a receptor, and the instant specification does not define “at least a portion”. Therefore, the claims are directed towards a chimeric peptide comprising a plurality of heterologous nuclear export domains linked to any portion of LAT.
Regarding claims 166, 168, 175, 177, Ho et. al. teaches a chimeric polypeptide comprising SEQ ID NO: 175, which comprises two NES of the sequence LALKLAGLDI flanking the amino acid sequence GGSGGS. As evidenced by Uniprot O43561 LAT_Human, the sequence GGS is residues 89-91 of the canonical human LAT isoform, and is therefore a portion of a LAT.
Regarding claim 167, the examiner notes that no relationship between the chimeric polypeptide and the receptor is claimed, and therefore the broadest reasonable interpretation of the claim is a chimeric polypeptide comprising a plurality of heterologous nuclear export signal domains linked to an adaptor protein of a TCR or CAR, wherein the adaptor protein comprises at least a portion of LAT. As evidenced by Lo et. al., LAT is an adaptor protein of the T cell receptor.
Regarding claim 185, Ho et. al. SEQ ID NO: 175 is comprised by a polypeptide that is directed to the nucleus of a host cell (Abstract, [127-130]).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 166, 167, 170-172, 174, 181, 182, 183, and 185 are rejected under 35 U.S.C. 103 as being unpatentable over U.S. 20190345220 to Qi et. al. effectively filed 11 January 2016 in view of Baeumler, Toni A., et. al. "Engineering