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 .
DETAILED ACTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/6/2025 has been entered. Claims 1, 8, and 10 were amended.
Claims 1-2, 4, and 8-23 are pending in the instant application.
Priority
This application is a 371 of PCT/US2019/017630, filed on 2/12/2019 which claims priority to the provisional application 62629139 filed on 2/12/2018.
Claim Interpretation: ITAM Naming and Crippling
The CD3ζ unit of a chimeric antigen receptor (CAR) contains a total of six unique ITAMs. Within the CD3 unit of the T cell Receptor (TCR), there is one ITAM in each of the γ-, δ-, and ε- chains, and there are three ITAMs within the ζ-chain, shown below (Love, pg 3, col 1, para 1, doi: 10.1101/cshperspect.a002485). Importantly, the CD3ζ chain of the CAR is the same as the chain called “TCRζ” in the TCR. The ITAMs themselves comprise the shared motif of YXXL/I-X6-8-YXXL/I, wherein Y=tyrosine, L=leucine, I=isoleucine, and X=any amino acid shown below (Love, Fig 1).
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In the instant application, applicant refers to a “pair” of ITAMs, such as “ITAM1/2”. Instant Fig 8, shows the following diagram, listing ITAM1-6 within the CD3-zeta domain.
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For the purposes of claim interpretation, ITAM1 and ITAM2 were interpreted as being equivalent to the sequences YNEL and YDVL of TCRζ-1, shown by Love above. Similarly, ITAM3 and ITAM4 were interpreted as being equivalent to the sequences YNAL and YSEI of TCRζ-2; and ITAM5 and ITAM6 were interpreted as being equivalent to the sequence YQGL and YDAL of TCRζ-3.
Phosphorylation of the tyrosine residues (Y) mediates TCR signal transduction (Love, pg 3, col 2, para 1). “Crippled” ITAMs are those which lack one or more tyrosine residues required for signal transduction. It is known that a TCR with crippled TCRζ-1, TCRζ-2, and TCRζ-3 is still capable of TCR signaling because the three ITAMs within the CD3 unit are still preserved.
“It thus came as a surprise to find that a TCR deprived of functional CD3-ζ ITAMs and its wild-type counterpart had similar capacity for differential responsiveness (for instance, using the weak agonist L3V at 10-7 M, both ζa1- b1- c1- and P14 T cells readily upregulated CD25 an CD69” (Ardouin, doi: 10.1016/s1074-7613(00)80041-2; pg 416, col 2, para 1).
Crippling of the ITAMs has been performed as a means of prolonging T cell life, via dampening the amount of TCR signaling.
Regulator T cells (a.k.a. Treg or CD4+ T cells), upon stimulation of the TCR, carry out immunosuppressive functions (e.g. via consuming inflammatory IL-2, producing immunosuppressive molecules, and suppression of antigen-presenting cells). There is an upper and lower limit of TCR stimulation required by Tregs in order for them to persist. Upon overstimulation, they can undergo Activation Induced Cell Death (AICD) (Zhao, J Immunol. 2009 Nov 1;183(9):5563-74). Upon understimulation, Tregs undergo a process called “death by neglect” (Togashi, Fig 1, doi: 10.1038/s41571-019-0175-7).
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.
Claims 1-2, 4, and 8-23 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.
claim 1, 10
Claims 1 and 10 are drawn to the CD3 zeta domain of a CAR comprising “a crippled ITAM pair … wherein the crippled ITAM pair is selected from the group consisting of ITAM1/2 and ITAM5/6”. It is unclear how this claim limitation is to be satisfied. Please see the “Claim Interpretation” section above as the nomenclature used is confusing. For the purposes of explanation, the native TCRζ-1 sequence (a.k.a. CD3-zeta sequence of the CAR that contains the underlined ITAMs 1 and 2) is:
NQL-YNELNLGRREE-YDVL
In Interpretation (I), both pairs of ITAMs 1 and 2 need to be mutated, thus the TCRζ-1 sequence of NQL-FNELNLGRREE-FDVL satisfies the claim limitation, but NQL-YNELNLGRREE-FDVL does not.
In Interpretation (II), only one member of the pair of ITAM1/2 needs to be mutated, thus the TCRζ-1 sequence of NQL-YNELNLGRREE-FDVL does satisfy the claim limitation.
Because these different interpretations lead to conflicting results, these claims are rendered indefinite. The analogous interpretations of “ITAM5/6” also render the claims indefinite for the same reasons.
Dependent claim 18 is drawn to the CD3-zeta domain comprising SEQ ID NO: 1 or SEQ ID NO: 2, wherein SEQ ID NO: 1 comprises mutated ITAM 1 and 2, whereas SEQ ID NO: 2 comprises no mutations at all, thus fails to cure these deficiencies. See 112(d) rejection of claim 18.
Dependent claims 2, 4, 8-9, 11-17, and 19-23 also fail to cure these deficiencies, thus are also rendered indefinite.
Claim Rejections – 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 11-13 and 18 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 11
Claim 11 is drawn to a Treg expressing the CAR of claim 10, however parent claim 10 already establishes that the CAR is expressed in a Treg, thus claim 11 fails to further limit the scope of parent claim 1.
Claim 12
Claim 12 is drawn to a Treg expressing the CAR of claim 1, however parent claim 1 already establishes that the CAR is expressed in a Treg, thus claim 12 fails to further limit the scope of parent claim 1.
Claim 13
Claim 13 is drawn to a Treg expressing the CAR of claim 4, however parent claim 4 already establishes that the CAR is expressed in a Treg, thus claim 13 fails to further limit the scope of parent claim 4.
Claim 18
Claim 18 is drawn to the CD3-zeta sequence comprising either SEQ ID NO: 1 or 2. Notably, SEQ ID NO: 2 comprises no mutated ITAMs, thus represents an expansion of scope over parent claim 1. The amino acid translation of instant SEQ ID NOs: 1 and 2 is shown below with ITAMs1-6 underlined, and the mutated residues within those ITAMs has been made in bold.
>instant_1
RAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGV
YNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPR
>instant_2
RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGV
YNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Allowable Subject Matter
This allowable subject matter section is predicated on this interpretation of the Figures described in the Declaration submitted on 6/5/2025 (wherein * indicates a mutated residue within the underlined ITAM according to claim 1):
ITAM1- comprises mutated ITAM 1 and 2:
TCRζ-1 NQL-*NELNLGRREE-*DVL
ITAM2- comprises mutated ITAM 5 and 6:
TCRζ-3 DGL-*QGLTSATKDT-*DAL
ITAM1-3- comprises mutated ITAM 1, 2, 5, and 6:
TCRζ-1 NQL-*NELNLGRREE-*DVL
TCRζ-3 DGL-*QGLTSATKDT-*DAL
The option of instant SEQ ID NO: 1 for claim 18 would be rendered allowable in light of surprising results (and upon correction of the 112 rejections of claims 1 and 18). Instant SEQ ID NO: 1 encodes a CD3 region comprising mutated ITAMS 1 and 2, as shown below.
RAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGV
YNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPR
Fig 1 of the declaration shows mutating (i) ITAM 1 and 2; (ii) ITAM 5 and 6; or (iii) ITAMs 1, 2, 5, and 6 does not affect Treg viability in the presence of FVIII. In other words, the CAR does not cause the Tregs to be overstimulation into cell death via AICD upon FVIII stimulation. Interestingly, the CARs actually prevent AICD over wildtype, which in itself is a surprising result.
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Fig. 2 of the declaration shows that downstream reporters of TCR signaling, Ki67 and CD69 are maintained in mutants (i) and (ii), however the mutant (iii) maintains wild-type levels of downstream signaling in the presences of FVIII despite having four crippled ITAMs. This is surprising considering mutant (iii) is able to exhibit similar levels of signaling and reduced cell death.
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Rana (doi: 10.1016/j.ymthe.2021.04.034) states “Approaches for tapering the magnitude of CAR signaling, such as introducing targeted mutations in CD3ζ ITAMs, have been carried out previously, with some success, although this strategy has not been tested for CAR Tregs. A decrease in the number of ITAM pairs from three to two or even one in the CAR construct can increase selectivity and prevent off-target effects by increasing the activation threshold. Our studies show that although Tregs are inherently more resistant to [Activation Induced Cell Death] AICD, introducing the ITAM1− mutation in CD3ζ (CD247) improved CAR Treg persistence in vivo. Interestingly, Tregs are reported to preferentially express the alternatively spliced θ isoform of CD247, which naturally lacks the ITAM3 domain, rather than the CD3ζ isoform” (pg 2688, col 1, para 3).
In the above quotation, Rana establishes that (1) mutating ITAMs has led to unpredictable results regarding their success in eliminating AICD in cytotoxic T cells and TILs; and (2) this 2021 publication above is the first publication of combining the crippled ITAMs within Tregs, which was co-authored by the instant inventors Herzog, Biswas, and Brusko after the priority date of the instant application.
Absent the disclosure of the instant application, one of skill in the art would have no reasonable expectation of success that crippling
ITAMs 1 and 2;
ITAMs 5 and 6; or
ITAMS 1, 2, 5, and 6,
would result in reduced AICD and wild-type or greater levels of Treg activation.
Response to Arguments
Applicant's arguments filed 10/6/2025 have been fully considered but they are not persuasive.
103; pg 5, para 2-pg 6, para 2
Applicant repeats the argument about Cambier’s teaching C-terminal truncation of TCRζ-1 NQLYNELNLGRREYDVL to NQLYNELNLGRR ablated TCR signaling activity in T effector (Teff) cells as evidence of the cited reference being inoperable. Applicant argues that Juilerat fails to produce an activated T cell.
Importantly, cell type is critical to the prosecution of the instant application. Treg cells suppress Teff cells, thus the teachings of Cambier which are drawn solely to Teff cells, no longer apply to the instantly claimed invention which is now limited to Treg cells. The cells of Julierat are also immunostimulatory, killer T cells, thus this art is no longer relevant to the discussion of the effects on Tregs. As an aside, Juilerat teaches their N-CAR with crippled ITAMs produces a less-active T cell, as opposed to an TCR inactive T cell as applicant appears to be implying.
103; pg 6, para 3-pg 7, para 1
Applicant argues (i) Zhao teaches when ITAM 1, 2, 5, and 6 (Zhao ITAM A and C) were mutated, the T cells were less prone to apoptosis; and (ii) Zhao teaches ITAM 1, 2, 5, and 6 transmit a stronger apoptosis signal over ITAM 3 and 4 (Zhao ITAM B); thus (iii) ITAM 1, 2, 5, and 6 are required to inhibit apoptosis. Applicant argues “Applicant reiterates that Zhao establishes a strong inference that costimulation of transformed cells with 4-lBB is necessary for T-cell activation.” Applicant correctly argues that the skilled artisan would not know what the effects of mutating one or more ITAMs in CAR expressed in a Treg.
The fact pattern of (i) and (ii) is not consistent with establishing a requirement that ITAMs 1, 2, 5, and 6 must be mutated in order to avoid apoptosis. The evidence of Zhao, as quoted by applicant, merely weights ITAMs 1, 2, 5, and 6 as having a stronger anti-apoptotic effect than ITAMs 3 and 4. Applicant did not provide evidence of this strong inference, nor is the record clear on when applicant had previously made this assertion. This assertion is further complicated by applicant’s invention also including a 4-1BB domain (claim 22), establishing that applicant agrees with the references that a 4-1BB domain is at the very least, beneficial, towards generating activated Tregs. The reference of Zhao is rendered irrelevant due to being drawn to PBLs and not Tregs.
103; pg 7, para 2
Applicant brings up a confounding finding in the reference of Zhao, where mutation of particular ITAMs leads to differences in the ability of the PBLs to produce the pro-apoptotic signals in the tumor cell lines.
It is recognized that the nomenclature used across publications is inconsistent, creating confusion. Importantly (i) Zhao’s 28Z CAR comprises zero mutated ITAMs; (ii) Zhao’s 28ZM lacks all ITAMs 1-6; (iii) and Zhao’s 4D5-28D lacks the entirely CD3ζ domain, entirely. In essence, this argument erroneously confuses the pro-apoptotic signals in the cancer cells with the pro-apoptotic signals within the PBLs. Thus applicant erroneously concludes that the mutations of the ITAMs leads to increased apoptosis of the PBLs themselves, however this is not what Zhao is stating. Zhao is saying that the CAR-containing PBLs were more effective in treating cancer than PBLs without the CAR. More pertinently, because the publication of Zhao is drawn to Peripheral Blood Lymphocytes (PBLs), it is no longer relevant to the discussion of the newly added limitation of the cells being Tregs. However, the finding of Zhao that the 28ZM CAR resulted in same amount of cancer killing as the control cells shows that in stimulatory T cells, the crippling of the ITAMs lowers the ability of the cells to kill cancer.
103; pg 7, para 3
Applicant points to instant Fig 1 of the declaration as showing mutating (i) ITAM 1 and 2; (ii) ITAM 5 and 6; or (iii) ITAMs 1, 2, 5, and 6 does not affect Treg viability in the presence of FVIII.
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Applicant points to Fig. 2 of the declaration, that downstream reporters oof TCR signaling, Ki67 and CD69 are maintained in mutants (i) and (ii). Applicant has appeared to identify “position A” with “ITAM 1-” and “position C” with “ITAM 3-.” As presented in arguments 8-11 of the declaration.
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Examiner greatly appreciates the footnote describing that “ITAMS1-3-” refers to mutating ITAM pairs ITAM1/2 and ITAM5/6. However, as described in the 112(b) rejection above, it is unclear if this mutant contains mutations at ITAMs 1, 2, 5, and 6; or if it contains only mutations at ITAMs 1 and 5. Upon clarification of what this mutation is, this indeed is a surprising result in the context of Treg cells. Fig 2 shows that mutating (presumably) (i) ITAMs 1 and 2; or (ii) ITAMs 5 and 6; in a Treg cell results in a population of activated Tregs with reduced apoptosis, but very interestingly, the combination of mutations (i.e. mutating ITAMs 1, 2, 5, and 6) does not yield activated Tregs. Absent the disclosure of the instant application, one of skill in the art would not have been able to predict this result given the art of record. Clarification of the mutations described in Fig 2 is required in order to arrive at a patentable invention.
Conclusion
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/L.A.E./
Examiner, Art Unit 1675
/JEFFREY STUCKER/Supervisory Patent Examiner, Art Unit 1675