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 .
1. The Election filed April 2, 2026, in response to the Office Action of December 4, 2025, is acknowledged and has been entered. Applicants elected without traverse Group I and the species of:
A. IgG Fc region mutation E269R and E294K; and
B. K131D and N74Q, and SEQ ID NO:88.
All species of Fc and CD16A mutations and SEQ ID NOs have been rejoined or examination.
Claims 1, 2, 5, 9, 12, 13, 19, 25-28, 34, 40, 41, 47, 99-105 are now pending. Claims 12, 13, 25, 19, 102, and 103 have been withdrawn from further consideration by the examiner under 35 CFR 1.142(b) as being drawn to non-elected inventions. It is noted that Applicants state claim 105 is encompassed by the elected invention, however, claim 105 is dependent upon a non-elected claim 103. It appears this may be a typo, given the claim language is directed to a composition, and Examiner has included claim 105 with the elected invention, addressing the claim dependency issue below. Claims 1, 2, 5, 9, 26-28, 34, 40, 41, 47, 99-101, 104, and 105 are currently under prosecution.
Broadest Reasonable Claim Interpretation
2. Claim 2 recites: “wherein the wild type or naturally occurring CD16A comprises an amino acid sequence shown in SEQ ID NO:78”. Claim 2 is reasonably interpreted for CD16A to encompass any sequences as small as two consecutive amino acids long that are “shown in SEQ ID NO:78”.
Claim 5 recites: “(d) wherein the CD16A comprising at least one amino acid mutation comprises an amino acid sequence shown in SEQ ID NO:80, 82, 84, 86, or 88”.” Claim 5 is reasonably interpreted for CD16A to encompass any sequences as small as two consecutive amino acids long that are “shown in SEQ ID NO:80” or the other listed SEQ ID NOs.
Claims 101 and 105 recite: “(d) wherein the non-naturally occurring CD16A comprising at least one amino acid mutation comprises an amino acid sequence shown in SEQ ID NO:80, 82, 84, 86, or 88”. Claims 101 and 105 are reasonably interpreted for CD16A to encompass any sequences as small as two consecutive amino acids long that are “shown in SEQ ID NO:80” or the other listed SEQ ID NOs.
Claim Rejections - 35 USC § 112
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.
3. Claim 105 is 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 105 depends from claim 103 and recites the limitation "the pharmaceutical composition of claim 103". There is insufficient antecedent basis for this limitation in the claim because there is no pharmaceutical composition recited in claim 103 or the claims it depends from. For the sake of compact prosecution, it is presumed that claim 105 was intended to depend from claim 104 and will be examined as such.
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.
4. Claims 1, 2, 5, 9, 26-28, 34, 40, 41, 47, 99-101, 104, and 105 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 pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a WRITTEN DESCRIPTION rejection.
The claims are drawn to a polypeptide comprising a modified Fc region of IgG, wherein the modified Fc region is non-naturally occurring and comprises at least one amino acid mutation compared to an Fc region of a wild type or naturally occurring IgG, and the polypeptide has essentially no binding activity to a wild type or naturally occurring Fcγ receptor and is capable of binding to a non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to the wild type or naturally occurring Fcγ receptor.
No sequence structure is recited for the modified Fc region of IgG that is required to function by:
having essentially no binding activity to a wild type or naturally occurring Fcγ receptor; and
capable of binding to a non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to the wild type or naturally occurring Fcγ receptor.
Dependent claims describing the Fcγ receptor to which the polypeptide binds provide no information on the structure of the polypeptide comprising a modified Fc region of IgG.
Although claim 9 recites in part (b) that the polypeptide is an antibody, requiring the antibody function by binding to a cancer antigen, no sequence structure is recited for the modified Fc region to perform the claimed functions.
Although claim 9 recites that the polypeptide comprises a modified Fc region of Igγ1 and comprises (a) mutation E269R, and one of mutations E294R or E294K, claim 9 does not provide the sequence structure of the “modified Fc region of Igγ1” comprising one or more amino acid mutations. With regards to a modified Fc region of IgG, the instant specification discloses:
[0096] The polypeptide comprises an Fc region of IgG. In some embodiments, the Fc region of IgG is the Fc region of human IgG, and may contain a polymorphism. In some embodiments, the Fc region of IgG is the Fc region of human Igγ1. The amino acid sequence of the Fc region can be easily obtained by those skilled in the art from a public database such as UniProt. The polypeptide of the present invention may be any form of polypeptide as long as it contains an Fc region of IgG. For example, a polypeptide of the present invention may be an antibody and an Fc fusion protein of a biomolecule or a fragment thereof and an Fc region.
[0098] In some embodiments, the Fc region is a modified Fc region of IgG, and the Fc region is non-naturally occurring and comprises at least one amino acid mutation compared to an Fc region of a wild type or naturally occurring IgG. In the present invention, “amino acid mutation” in the Fc region refers to a substitution, deletion, or insertion of an amino acid at a predetermined amino acid position in the Fc region.
Therefore, according to the specification, the claims encompass a vast genus of IgG or Igγ1 (IgG1) Fc regions having any modifications, including any number of unknown substitutions, deletions, or insertions of an amino acid at any position in the Fc region, according to the specification. Therefore the claims encompass a vast genus of modified Fc regions of IgG or Igγ1 (IgG1) having unlimited modifications or amino acid mutations, wherein the Fc region is required to perform the functions claimed and listed above.
Claims 41, 47, 99, 100, 101 are also drawn to a kit comprising a cell expressing a non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to a wild type or naturally occurring Fcy receptor, wherein the polypeptide is capable of binding to the non-naturally occurring Fcy receptor comprising at least one amino acid mutation.
No sequence structure is recited for the non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to a wild type or naturally occurring Fcy receptor to perform the function of:
capable of binding to the non-naturally occurring Fcγ receptor comprising at least one amino acid mutation.
Dependent claim 100 recites that the non-naturally occurring Fcγ receptor is a non-naturally occurring CD16A comprising at least one amino acid mutation; and/or CD16A comprises at least one mutation selected from K131D mutation, K128E mutation, and K131E mutation relative to SEQ ID NO:78. The specification discloses:
III. Cells Expressing CD16A Comprising Amino Acid Mutations
[0119] The present invention also provides a CD16A-expressing cell that can be used in combination with a polypeptide of the present invention.
[0120] In one aspect, the present invention provides a cell expressing a non-naturally occurring CD16A comprising at least one amino acid mutation compared to a wild type or naturally occurring CD16A, wherein the at least one amino acid mutation is selected from (i) a lysine to an aspartic acid at a position corresponding to position 131 in SEQ ID NO: 78 (K131D mutation), (ii) a lysine to a glutamic acid at a position corresponding to position 128 in SEQ ID NO: 78 (K128E mutation), and (iii) a lysine to a glutamic acid at a position corresponding to position 131 in SEQ ID NO: 78 (K131E mutation), and wherein the non-naturally occurring CD16A comprises an amino acid sequence having 90% or more amino acid sequence identity with SEQ ID NO: 78.
[0122] In an embodiment of the present invention, the CD16A expressed by a CD16A-expressing cell of the present invention comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 78.
Thus, claim 100 is encompasses a vast genus of CD16A or SEQ ID NO:78 variants comprising any number of unknown mutations, as well as additional mutations K131D, K128E, or K131E.
Dependent claims 41, 47, 100, and 101 do not define the sequence structure of the non-naturally occurring Fcγ receptor or CD16A that is required to perform the claimed functions of binding to the non-naturally occurring Fcy receptor comprising at least one amino acid mutation.
It is noted that claim 27 recites a pharmaceutical composition comprising the polypeptide according to claim 1 “for combined use with a cell for immunotherapy, wherein the cell expresses a non-naturally occurring Fcy receptor comprising at least one amino acid mutation compared to a wild type or naturally occurring Fcy receptor, wherein the polypeptide is capable of binding to the non-naturally occurring Fcy receptor comprising at least one amino acid mutation”, therefore claim 27 recites an intended use of the polypeptide in combination with the cell, but does not require the cell expressing the non-naturally occurring Fcy receptor to be included or present in the pharmaceutical composition. However, claim 27 encompasses the vast genus of polypeptides comprising modified Fc regions, as stated above.
With regard to representative species of modified Fc region of IgG that function as claimed, the instant specification discloses:
[0098] In an embodiment of the present invention, the Fc region is the Fc region of human Igγ1, and the amino acid mutation in the Fc region is an amino acid mutation in the one or two amino acid positions selected from the amino acids in amino acid positions 269 and 294 within the human Igγ1 constant region according to EU index numbering. In an embodiment of the present invention, the human Igγ1 constant region prior to introduction of a mutation contains the amino acid sequence shown in SEQ ID NO: 24. In an embodiment of the present invention, the human Igγ1 Fc region prior to introduction of a mutation contains sequence 1 to 330 in the amino acid sequence shown in SEQ ID NO: 24.
[0106] As used herein, the phrase “having essentially no binding activity” to a wild type Fcγ receptor refers to the binding activity of a polypeptide of the present invention to a wild type Fcγ receptor is not significantly higher than the binding activity of the polypeptide of the present invention to the Fcγ receptor containing at least one amino acid mutation to which the polypeptide of the present invention binds. In an embodiment of the present invention, when “having essentially no binding activity” to a wild type Fcγ receptor is measured using the ELISA method, this is 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less, or 0.05% or less of the binding activity to the Fcγ receptor containing at least one amino acid mutation used as the control.
With regard to representative species of non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to a wild type or naturally occurring Fcγ receptor that function as claimed, the instant specification discloses:
[0107] The Fcγ receptor targeted by a polypeptide of the present invention can be selected by those skilled in the art based on the intended use for the polypeptide of the present invention and other factors. In an embodiment of the present invention, the Fcγ receptor can be FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), or FcγRIIIB (CD16B). In an embodiment of the present invention, the Fcγ receptor targeted by a polypeptide of the present invention is CD16. In an embodiment of the present invention, the Fcγ receptor is CD16A. Wild type CD16A includes, but not limited to, two polymorphisms: CD16A V158 and CD16A F158. In an embodiment of the present invention, the wild type CD16A is CD16A V158.
[0108] In an embodiment of a polypeptide of the present invention, the target Fcγ receptor is CD16A and the wild type CD16A contains the amino acid sequence shown in SEQ ID NO: 78.
[0109] In an embodiment of a polypeptide of the present invention, the target Fcγ receptor is CD16A comprising at least one amino acid mutation, and the CD16A comprising at least one amino acid mutation comprises at least one mutation selected from a mutation from the lysine to the aspartic acid at a position corresponding to position 131 in SEQ ID NO: 78 (K131D mutation), a mutation from the lysine to the glutamic acid at a position corresponding to position 128 in SEQ ID NO: 78 (K128E mutation), and a mutation from the lysine to the glutamic acid at a position corresponding to position 131 in SEQ ID NO: 78 (K131E mutation). In an embodiment of the present invention, the CD16A comprising at least one amino acid mutation contains one or both of the K131D mutation and the K128E mutation. In an embodiment of the present invention, the CD16A comprising at least one amino acid mutation comprises one or both of the K131E mutation and the K128E mutation. In an embodiment of the present invention, the CD16A comprising at least one amino acid mutation comprises the K131D mutation and at least one mutation selected from a mutation from the asparagine to the glutamine at a position corresponding to position 38 in SEQ ID NO: 78 (N38Q mutation) and a mutation from the asparagine to the glutamine at a position corresponding to position 74 in SEQ ID NO: 78 (N74Q mutation).
[0111] In an embodiment of the present invention, the CD16A comprising at least one amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, or SEQ ID NO: 88.
With regards to representative species of polypeptides comprising modified Fc regions that bind to non-naturally occurring Fcγ receptor and have essentially no binding to wild type or naturally occurring Fcγ receptor, and the species of non-naturally occurring Fcγ receptor or CD16A comprising at least one amino acid mutation compared to wild type or naturally occurring Fcγ receptor that the modified Fc region binds, the specification discloses:
EXAMPLES
[0196] There are two polymorphisms of the human CD16A: CD16A V158 and CD16A F158. The following examples were performed using CD16A V158 (“CD16V” below) which has higher binding activity to antibody Fc. The experiments using commercial kits or regents have been performed by following the attached protocol except when the method was demonstrated.
Example 1: Protein Design Using In Silico Calculations
[0197] It has been reported by analyzing the three-dimensional structure of a complex protein that the binding activity and stability of a complex are affected by the introduction of a mutation into a charged amino acid that is considered important to complex formation, and that in silico calculations can be used to predict the effect of introducing mutations to charged amino acids on binding activity (Scientific Reports (2019) 9, pp. 4482). Therefore, the present inventors analyzed the three-dimensional structure of a complex between CD16V and antibody Fc (PDB code; 3ay4) using the MOE software (Chemical Computing Group), and extracted the basic or acidic amino acid residues on the binding interface between the CD16V and antibody Fc. Among these amino acid residues, a mutant was designed in which a basic amino acid on CD16V was replaced with an acidic amino acid or an acidic amino acid on the antibody Fc was replaced with a basic amino acid (Table 1). The names of the CD16V mutant described in the following example were in accordance with the number of the amino acid residues of the CD16A protein registered in 3ay4, which is based on the sequence of the CD16V protein (GenBank accession number: AAH17865.1) excluding the 1st to 18th amino acid sequences (SEQ ID NO: 78) (“3ay4” in the table). The mutations in the CD16V protein containing the 1st to 18th amino acid sequence corresponding to the mutations shown in “3ay4” are shown in the column “AAH17865.1” in Table 1.
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Example 2: Preparation of a CD16V Protein and CD16V Mutant Proteins
[0199] In the present example, a protein with a mutation introduced to CD16V is denoted by “CD16V_introduced mutation” and referred to below collectively as CD16V mutant.
[0200] In order to obtain a CD16V protein, a gene encoding a polypeptide in the extracellular portion of CD16V (amino acids 1 to 208 in GenBank accession number: AAH17865.1) in which a FLAG sequence (DYKDDDDK, SEQ ID NO: 91) is linked to the C-terminus (SEQ ID NO: 1) was subcloned into a pcDNA3.4 vector (Thermo Fisher Scientific, Cat. A14697). The constructed vector was then transfected into ExpiCHO-S cells (Thermo Fisher Scientific, Cat. A29133). In order to obtain CD16V mutant proteins, a gene encoding a polypeptide in the extracellular portion of CD16V with an amino acid mutation shown in Table 1 (CD16V_K120D, CD16V_K120E, CD16V_K128D, CD16V_K128E, CD16V_K131D, CD16V_K131E, CD16V_K161D or CD16V_K161E) and a FLAG sequence linked to the C-terminus (SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17) was introduced to a pcDNA3.4 vector. The constructed vector was then transfected into ExpiCHO-S cells. The CD16V protein and the CD16V mutant protein were purified from the culture supernatant of ExpiCHO-S cells according to a standard method using anti-FLAG (registered trademark) M2 antibody affinity gel (SIGMA-ALDRICH, Cat. A2220). Note that the 1st to 18th amino acid sequences in the CD16V of AAH17865.1 are cleaved in mature form. The positions of the amino acid mutations introduced into the CD16V protein described in this example are in accordance with the amino acid numbers registered in 3ay4, and are numbered based on CD16V excluding the 1st to 18th amino acid sequence. Each mutation in the amino acid sequence of each CD16V mutant protein described in the sequence listing corresponds to a mutation shown in the “AAH17865.1” column of Table 1.
Example 3: Production of an Fc_wt-Type Anti-HER2 Antibody or Mutant Fc-Type Anti-HER2 Antibodies
[0201] In the following study, trastuzumab (Drug Bank Accession Number: DB00072) was used as the anti-HER2 antibody.
[0202] Antibodies having the Fc sequence of the wild type human Igγ1 constant region are collectively referred to as Fc_wt. An expression vector used for production of a Fc_wt-type anti-HER2 antibody was constructed in the following manner. A heavy chain expression vector was constructed by inserting into a pcDNA3.4 vector a polynucleotide of a gene encoding the heavy chain variable region of trastuzumab (SEQ ID NO: 21) with a gene encoding a signal sequence (MEFGLSWVFLVAILKGVQC) (SEQ ID NO: 19) added to the 5′-side and a gene encoding the human Igγ1 constant region (SEQ ID NO: 23) added to the 3′-side. A light chain expression vector was constructed by inserting into a pcDNA3.4 vector a polynucleotide of a gene encoding the light chain region of trastuzumab (SEQ ID NO: 27) with a gene encoding a signal sequence (MDMRVPAQLLGLLLLWLRGARC) (SEQ ID NO: 25) added to the 5′-side. This light chain expression vector is referred to below as the trastuzumab light chain expression vector. These vectors were co-transfected into ExpiCHO-S cells, and an anti-HER2 antibody having a wild type Fc region (referred to below as the Fc_wt-type) was prepared from the culture supernatant according to a standard method.
[0203] Antibodies having a mutation in the Fc region (referred to below as “Fc_introduced mutations” and referred to below collectively as mutant Fc-type antibodies) were prepared.
[0204] Heavy chain expression vectors used in the production of Fc_S239K, Fc_S239R, Fc_E294K, and Fc_E294R were constructed by introducing into pcDNA3.4 vectors a polynucleotide of a gene encoding the heavy chain variable region of trastuzumab (SEQ ID NO: 21) with a gene encoding a signal sequence (MEFGLSWVFLVAILKGVQC) (SEQ ID NO: 19) added on the 5′-side and with a gene encoding a human Igγ1 constant region into which amino acid mutations for substituting lysine (K) or arginine (R) at S239 or E294 have been introduced (SEQ ID NOs: 33, 35, 29 and 31) added on the 3-'side. Heavy chain expression vectors used in the production of Fc_D265K, Fc_D265R, Fc_E269K, and Fc_E269R were constructed by introducing into pcDNA3.4 vectors a polynucleotide of a gene encoding the heavy chain variable region of trastuzumab (SEQ ID NO: 39) with a gene encoding a signal sequence (MEWSWVFLFFLSVTTGVHS) (SEQ ID NO: 37) added on the 5′-side and with a gene encoding a human Igγ1 constant region into which amino acid mutations for substituting lysine (K) or arginine (R) at D265 or E269 have been introduced (SEQ ID NOs: 41, 43, 45 and 47) added on the 3-'side. Each one of these expression vectors was co-transfected with the trastuzumab light chain expression vector into ExpiCHO-S cells, and mutant Fc-type anti-HER2 antibodies were prepared from the culture supernatant according to a standard method.
Example 4: Fc-Type Anti-HER2 Antibodies With CD16V and CD16V Mutant Proteins
[0205] The binding activity of the CD16V and CD16V mutants obtained in Example 2 with the Fc_wt-type and mutant Fc-type anti-HER2 antibodies obtained in Example 3 was evaluated.
[0206] As a result, CD16V_K131D did not bind to the Fc_wt, but only to Fc_E269R. However, the binding activity between CD16V_K131D and Fc_E269R was lower than the binding activity between Fc_wt and CD16V. Also, Fc_E269R did bind slightly to CD16V. While it was confirmed that both CD16V_K128E and K131E did bind to mutant Fc, binding to Fc_wt was only somewhat confirmed. Meanwhile, no binding or extremely low binding of CD16V_K128D, CD16V_K120D, CD16V_K120E, CD16V_K161D and CD16V_K161E with Fc_wt or any of the mutant Fc was confirmed (FIG. 1).
[0207] This suggests that position K128 and position K131 in CD16V as well as position E269 and position E294 in the Fc region of the antibodies are important amino acid residues for specific binding activity between CD16V mutants and mutant Fc type antibodies. Therefore, a study was conducted to obtain mutant Fc and CD16V mutants that only binds specifically each other into which specific amino acid mutations have been introduced using combinations of these mutations and combinations of these mutations with mutations known to increase binding activity.
Example 5: Production of Mutant Fc-Type Anti-HER2 Antibodies and CD16V Mutant Proteins
[0208] CD16V_K128E_K131D (referred to as CD16V_ED below) and CD16V_K128E_K131E (referred to as CD16V_EE below) were prepared by combining mutations of K128E and K131D or K131E with CD16V. Specifically, as in Example 2, an expression vector was constructed by introducing to a pcDNA3.4 vector a gene encoding an extracellular polypeptide of CD16V protein with these amino acid mutations linked to a FLAG sequence (SEQ ID NOs: 49 and 51) introduced at the C-terminus. This expression vector was then transfected into ExpiCHO-S cells. CD16V_ED and CD16V_EE were prepared from the culture supernatant of each in the same manner as Example 2.
[0209] It has been reported that mutagenesis of N38Q, N74Q or N169Q into CD16V slightly enhances the binding activity of Fc_wt (Journal of Biological Chemistry (2018) 293, pp. 16842-16850). CD16V_K131D (referred to below as CD16V_D) bound only to the Fc_E269R-type antibody, therefore, CD16V_K131D_N38Q (referred to below as CD16V_DQ1) and CD16V_K131D_N74Q (referred to below as CD16V_DQ2) in which CD16V_D is combined with N38Q or N74Q mutations were prepared. Specifically, expression vectors were constructed in the same manner as Example 2 by introducing into a pcDNA3.4 vector a gene encoding a polypeptide of an extracellular domain of the CD16V protein, in which one of these amino acid mutations is introduced, linked to a FLAG sequence (SEQ ID NOs: 53 and 55) is introduced to the C-terminal. Each expression vector was then transfected into ExpiCHO-S cells. CD16V_DQ1 and CD16V_DQ2 were prepared from the culture supernatant of each in the same manner as Example 2.
[0210] Fc_E269R_E294K (referred to below as Fc_RK) and Fc_E269R_E294R (referred to below as Fc_RR) type anti-HER2 antibodies were prepared. Specifically, expression vectors were constructed in the same manner as Example 3 by introducing into a pcDNA3.4 vector a polynucleotide of a gene encoding the heavy chain variable region of trastuzumab (SEQ ID NO: 39) with a gene encoding a signal sequence (MEWSWVFLFFLSVTTGVHS) (SEQ ID NO: 37) added to the 5′-side and with a gene encoding a human Igγ1 constant region having amino acid mutations introduced to arginine at position E269 and to lysine (K) or arginine (R) at position E294 (SEQ ID NOs: 57 and 59) added to the 3′-side. The resulting heavy chain expression vector and the trastuzumab light chain expression vector obtained in Example 3 were co-transfected to ExpiCHO-S cells, and mutant Fc-type anti-HER2 antibodies were prepared from the culture supernatant of each according to a standard method.
Example 6: Evaluation of the Binding Activity of Fc_wt-Type and Mutant Fc-Type Anti-HER2 Antibodies With CD16V and CD16V Mutant Proteins
[0211] The binding activity of CD16V_D obtained in Example 2 and CD16V_ED, CD16V_EE, CD16V_DQ1 and CD16V_DQ2 obtained in Example 5 with Fc_wt type anti-HER2 antibody obtained in Example 3, or Fc_RK or Fc_RR type anti-HER2 antibodies obtained in Example 5 was evaluated in the same manner as Example 4 (FIG. 2 ).
[0212] As a result, the Fc_wt type anti-HER2 antibody bound to CD16V but did not exhibit binding activity to CD16V_D, CD16V_ED, CD16V_EE, CD16V_DQ1 and CD16V_DQ2. Meanwhile, the Fc_RK or Fc_RR type anti-HER2 antibodies bound to the CD16V mutant but not to CD16V. Therefore, it is clear that the binding specificity of the mutant Fc-type antibody to the CD16V mutant was enhanced by introducing a mutation at position E294 into Fc_E269R. Also, the binding activity of CD16V_DQ1 and CD16V_DQ2 to Fc_RK and Fc_RR was almost the same as the binding activity of CD16V to Fc_wt. Therefore, it is clear that CD16V_DQ1 and CD16V_DQ2, in which additional mutations are introduced into CD16V_D, do not have enhanced Fc_wt binding activity but only have enhanced Fc_RR and Fc_RK binding activity.
Thus, the instant specification describes antibody Fc IgG1 region SEQ ID NO:24 having a mutation specifically at amino acid positions listed in Table 1; and the specification describes Fcγ receptor SEQ ID NO:78 that is high affinity human CD16A comprising mutation V158, having mutations specifically at amino acid positions listed in Table 1, wherein functionally:
CD16V_K131D did not bind to the Fc_wt, but bound to Fc_E269R; and
CD16V_K128E and K131E did bind to mutant Fc, binding to Fc_wt was only somewhat confirmed.
Fc_E269R_E294K (referred to as Fc_RK) bound to the CD16V mutants (CD16V_D, CD16V_ED, CD16V_EE, CD16V_DQ1 and CD16V_DQ2) but not to CD16V;
Fc_E269R_E294R (referred to as Fc_RR) bound to the CD16V mutants (CD16V_D, CD16V_ED, CD16V_EE, CD16V_DQ1 and CD16V_DQ2) but not to CD16V;
CD16V_D, CD16V_ED, CD16V_EE, CD16V_DQ1 and CD16V_DQ2 did not bind to wild type Fc IgG1; and
No binding or extremely low binding of CD16V_K128D, CD16V_K120D, CD16V_K120E, CD16V_K161D and CD16V_K161E with Fc_wt or any of the mutant Fc was confirmed, therefore the modified Fc IgG regions tested did not bind the non-naturally occurring CD16A mutants CD16V_K128D, CD16V_K120D, CD16V_K120E, CD16V_K161D and CD16V_K161E and did not function as claimed.
Therefore, with regards to structure-function correlation, the specification discloses only the species of modified Fc IgG1 SEQ ID NO:24 with mutations E269R, E269R + E294K, or E269R + E294R, as critical to binding only to the species of CD16A V158 (CD16V) SEQ ID NO:78 with mutations K131D (CD16V_D), K131D+N38Q (CD16V_DQ1), K131D+N74Q (CD16V_DQ2), K128E, K128E+K131D (CD16V_ED), or K128E+K131E (CD16V_EE), and having essentially no binding activity to wild type CD16A or CD16V.
The specification fails to disclose any structural sequence required of any other modified Fc regions of IgG or IgG1 comprising at least one amino acid mutation compared to an Fc region of a wild type or naturally occurring IgG to possess the function of:
having essentially no binding activity to a wild type or naturally occurring Fcγ receptor; and
capable of binding to a non-naturally occurring Fcγ receptor comprising at least one amino acid mutation compared to the wild type or naturally occurring Fcγ receptor.
The specification fails to disclose any structural sequence required of any other non-naturally occurring Fcγ receptor of CD16A comprising at least one amino acid mutation compared to wild type of naturally occurring Fcγ receptor to possess the function of:
capable of binding to the non-naturally occurring Fcγ receptor comprising at least one amino acid mutation;
and cells or immune cells expressing such non-naturally occurring Fcγ receptors.
To provide adequate written description and evidence of possession of the claimed Fc region and Fcγ receptor genera, the instant specification can structurally describe representative modified Fc regions of IgG and non-naturally occurring non-naturally occurring Fcγ receptors that function as claimed, or describe structural features common to the members of the genus, which features constitute a substantial portion of the genus. Alternatively, the specification can show that the claimed invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics (see University of California v. Eli Lilly and Co., 119 F.3d 1559, 43 USPQ2d 1398 (Fed. Cir. 1997) and Enzo Biochem, Inc. V. Gen-Probe Inc.).
In this case, the only factor present in the claims is a recitation of the Fc region and Fcγ receptor functions as listed above. The instant specification fails to describe structural features common to the members of the genus, which features constitute a substantial portion of the genus because the instant specification discloses only a single Fc IgG1 SEQ ID NO:24 with specific mutations at E269 with or without E294 mutations, and only a single CD16A V158 SEQ ID NO:78 with specific mutations at K131 and K128 that function binding together as claimed. A definition by function does not suffice to define the genus because it is only an indication of what the Fc region and Fcγ receptor do, rather than what they are. Other than for the species of Fc IgG1 SEQ ID NO:24 with specific mutations at E269 and E294 and CD16A V158 SEQ ID NO:78 with specific mutations at K131 and K128, the specification fails to provide the structural features coupled to the claimed functional characteristics for the vast genus of modified Fc regions and non-naturally occurring Fcγ receptors claimed. Applicants have not established any reasonable structure-function correlation with regards to the sequences in the Fc IgG region or Fcγ receptor or CD16A that can be altered and still maintain the claimed functions. The instant specification fails to describe a representative number of Fc region and Fcγ receptor sequences for the genus of Fc region and Fcγ receptor that function as claimed. Accordingly, in the absence of sufficient recitation of distinguishing identifying characteristics, the specification does not provide adequate written description of the claimed genus of modified Fc regions and non-naturally occurring Fcγ receptors.
Although Applicants may argue that it is possible to screen for Fc regions and Fcγ receptors that bind each other and function as claimed, the court found in (Rochester v. Searle, 358 F.3d 916, Fed Cir., 2004) that screening assays are not sufficient to provide adequate written description for an invention because they are merely a wish or plan for obtaining the claimed chemical invention. “As we held in Lilly, “[a]n adequate written description of a DNA … ‘requires a precise definition, such as by structure, formula, chemical name, or physical properties,’ not a mere wish or plan for obtaining the claimed chemical invention.” 119 F.3d at 1566 (quoting Fiers, 984 F.2d at 1171). For reasons stated above, that requirement applies just as well to non-DNA (or RNA) chemical inventions.” Knowledge of screening methods provides no information about the structure of any future Fc regions and Fcγ receptors yet to be discovered that may function as claimed. The Fc region provides no information about the structure of an Fcγ receptor that binds to it, and vice versa.
Given the lack of representative examples to support the full scope of the claimed Fc regions and Fcγ receptors, and lack of reasonable structure-function correlation with regards to the unknown sequences in the Fc regions and Fcγ receptors that provide the claimed functions, the present claims lack adequate written description. Thus, the specification does not provide an adequate written description of Fc regions and Fcγ receptors that bind to each other and function as claimed that are required to practice the claimed invention.
Claim Rejections - 35 USC § 102
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.
5. Claim(s) 1, 2, 5, 9, 26-28, 34, 40, 41, 99, 100, 101, 104, and 105 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by WO 2022/029051, Darowski et al, claiming priority to August 3, 2020.
Darowski teaches an antibody comprising a modified Fc IgG1 region comprising amino acid mutation P329G that does not occur in nature and causes the Fc region to be effector-silenced, meaning it has essentially no binding to naturally occurring IgG1 Fc receptor CD16A (p. 5, 25-28, 61-63; Table 7).
Darowski teaches the antibody binds to a cancer antigen (p. 5, 57-58, 68).
Darowski teaches the modified Fc IgG1 region is specifically recognized and bound by a CAR comprising: (i) a CD16A (FCGR3A) transmembrane domain and/or CD16A signaling domain, and (ii) a binding domain that comprises an antibody that specifically recognizes and binds to the P329 Fc mutation, therefore does not bind to wild-type Fc (Figure 1A; p. 1-2, 7, 24-28, 32-34, 39-42, claims 1-6). The CAR taught by Darowski is encompassed by the instantly claimed genus of non-naturally occurring CD16A comprising at least one amino acid mutation and binds to a modified region of IgG that is non-naturally occurring and comprises at least one amino acid mutation compared to an Fc region of a wild type IgG.
Darowski teaches the known sequence of CD16A (FCGR3A) is SEQ ID NO:88 (p. 34) which comprises 100% of instant SEQ ID NO:78 (see sequence alignment below), therefore the CAR of Darowski comprising the transmembrane and/or signaling domain of CD16A (FCGR3A) necessarily comprises “an amino acid sequence shown in” instant SEQ ID NO:78.
CD16A SEQ ID NO:88 of Darowski is 99.1% identical to instant SEQ ID NO:80 (see sequence alignment below), therefore the CAR of Darowski comprising the transmembrane and/or signaling domain of CD16A (FCGR3A) necessarily comprises “an amino acid sequence shown in” instant SEQ ID NO:80.
Darowski teaches an immune cell expressing the CAR, wherein the immune cell is a T cell (p. 2, 25-26, 49-53; claims 13, 21-24).
Darowski teaches kits comprising the antibody comprising a modified Fc IgG1 region and immune cells expressing the CAR (p. 2, 64-67; claims 16-20).
Darowski teaches pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the antibody comprising a modified Fc IgG1 region and/or immune cells expressing the CAR (p. 72-75; claims 20-22 and 25).
It is noted that in claim 27, the preamble recitation of “for combined use with a cell for immunotherapy” for the claimed pharmaceutical composition is merely suggestive of an intended use and is not given weight for purposes of comparing the claims with the prior art. The claims read on the active ingredients per se, which are the polypeptide of claim 1 and a pharmaceutically acceptable excipient (see MPEP 2111.02). Claims 28 and 34 describe the cell that is not included in the composition.
Instant CD16A SEQ ID NO:78 aligned with Darowski CD16A (FCGR3A) SEQ ID NO:88:
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462
622
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Greyscale
Instant CD16A SEQ ID NO:80 aligned with Darowski CD16A (FCGR3A) SEQ ID NO:88:
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536
628
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Greyscale
6. Conclusion: No claim is allowed.
7. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642
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