Office Action 5: Non-Final Rejection (2024-08-28)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
Amendments to the Claims:

This listing of claims will replace all prior versions, and listings, of claims in the application:

Listing of Claims:

Amdt date August 21, 2024 Reply to Office action of February 21, 2024

1 - 22. (canceled)221

23\. (Currently Amended) A preparation method of an antibody-drug conjugate with a structure as shown in formula I:mAb-(L-D)n I wherein,
mAb represents a recombinant anti-CD20 monoclonal antibody, wherein the recombinant anti-CD20 monoclonal antibody is rituximab or a biosimilar thereof;
D represents a small molecule toxin, wherein the small molecule toxin is monomethyl auristatin-E (MMAE);
L is a linker connecting the antibody and the small molecule toxin, wherein the linker is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl;n is an average number of the small molecule toxins conjugated to the antibody, and n is in a range of 4.2SVG {EMBED HERE} 0.100.080.5; and
"-"is a bond, and
wherein the structure of the antibody-drug conjugate is the following formula:mAb andSVG {EMBED HERE} 1.3836.06

wherein the preparation method comprises the steps of:
(1) performing a reduction reaction between the recombinant anti-CD20 monoclonal antibody and a reducing agent in a PB (phosphate buffer) reaction buffer (pH 7.6) at 25±10C for 90±10 minutes, thereby obtaining a reaction system comprising a reduced recombinant anti-CD20 monoclonal antibody, wherein the reducing agent is tris (2-carboxyethyl) phosphine, and a molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1: 2.9 to 1: 3.1;
(2) adding a solution of maleimidocaproyl-vc-PAB-MMAE in acetonitrile and water dropwise to the reaction system of step (1) and performing a coupling reaction at 4±0.5 0C for 60±10 minutes to obtain a coupling solution, wherein a volume ratio of acetonitrile to water is 1:1, and a molar ratio of the recombinant anti-CD20 monoclonal antibody in step (1) to the small molecule toxin is 1: 7.0 to 1: 8.0;
(3) adding into the reaction system obtained from step (2) a stopping solution of L-cysteine which is prepared by dissolving L-cysteine in a diethylenetriaminepentaacetic acid solution, to terminate the coupling reaction; and
(4) when the coupling reaction in step (3) is terminated, transferring the coupling solution to a tangential flow filtration (TFF) system for buffer replacement, thereby replacing the PB reaction buffer with a HT preparation buffer, and filtering the solution obtained with a 0.22 micrometer Rapid - Flow device to make a residual rate of the PB reaction buffer in a final solution less than 0.25%, wherein the HT preparation buffer contains 3.18 g of L-histidine, 70 g of trehalose dihydrate, and 0.2 mL of Tween 80 per L, with pH adjusted to 6.5 using 0.5 mol/L hydrochloric acid, and then filtered through a 0.22 pm membrane.

24\. (Previously Presented) The preparation method of claim 23, wherein n is in a range of 4.2 ±0.3.

25\. (Previously Presented) The preparation method of claim 23, wherein in step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is about 1:2.95-1:3.05.

26\. (Previously Presented) The preparation method of claim 23, wherein in step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is about 1:2.99-1:3.01.

27\. (Previously Presented) The preparation method of claim 23, wherein in step (1), the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is about 1:3.

28\. (Previously Presented) The preparation method of claim 23, wherein in step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody from step (1) to the small molecule toxin is about 1:7.2-1:7.7.

29\. (Previously Presented) The preparation method of claim 23, wherein in step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody from step (1) to the small molecule toxin is about 1:7.4-1:7.6.

30\. (Previously Presented) The preparation method of claim 23, wherein in step (2), the molar ratio of the recombinant anti-CD20 monoclonal antibody from step (1) to the small molecule toxin is about 1:7.5.

31\. (Previously Presented) The preparation method of claim 23, wherein n is 4.2, and in step (1), the reduction reaction between the recombinant anti-CD20 monoclonal antibody and the reducing agent is performed in the PB reaction buffer (pH

7.6) at 25 0C for 90 minutes, the molar ratio of the recombinant anti-CD20 monoclonal antibody to the reducing agent is 1:3; andin step (2), the solution of the small molecule toxin in acetonitrile and water is dropwise added to the reaction system of step (1) and the coupling reaction is performed at 4 0C for 60 minutes, and the molar ratio of the recombinant anti-CD20 monoclonal antibody in step (1) to the small molecule toxin is 1: 7.5.

32\. (Previously Presented) An antibody drug conjugate prepared according to the preparation method of claim 23, wherein the average number of the small molecule toxins conjugated to the antibody is 4.2 ± 0.5, and wherein components in the antibody drug conjugate ranked in descending order according to their contents are as follows: a component which has 4 small molecule toxins conjugated to the antibody (D4); a component which has 2 small molecule toxins conjugated to the antibody (D2); a component which has 6 small molecule toxins conjugated to the antibody (D6); a component which has 8 small molecule toxins conjugated to the antibody (D8); a the component which has 0 small molecule toxins conjugated to the antibody (DO).

33\. (Currently Amended) The antibody drug conjugate \[\[of\]\]accordinq to claim 32, wherein the average number of the small molecule toxins conjugated to the antibody is 4.2 ± 0.3, and wherein the components in the antibody drug conjugate ranked in descending order according to their contents are as follows: the component which has 4 small molecule toxins conjugated to the antibody (D4); the component which has 2 small molecule toxins conjugated to the antibody (D2); the component which has 6 small molecule toxins conjugated to the antibody (D6); the component which has 8 small molecule toxins conjugated to the antibody (D8); and the component which has 0 small molecule toxins conjugated to the antibody (DO).

34\. (Currently Amended) An antibody drug conjugate pharmaceutical preparation comprising an antibody drug conjugate prepared according to the preparation method of claim 23, wherein the average number of the small molecule toxins conjugated to the antibody is 4.2 0.5, and wherein components in the antibody drug conjugate pharmaceutical preparation ranked in descending order according to their contents are as follows: a component which has 4 small molecule toxins conjugated to the antibody (D4); a component which has 2 small molecule toxins conjugated to the antibody (D2); a component which has 6 small molecule toxins conjugated to the antibody (D6); a component which has 8 small molecule toxins conjugated to the antibody (D8); and a component which has 0 small molecule toxins conjugated to the antibody (DO).SVG {EMBED HERE} 0.100.08
35\. (Currently Amended) The antibody drug conjugate pharmaceutical preparation according to claim 34, wherein the average number of the small molecule toxins conjugated to the antibody is 4.2 SVG {EMBED HERE} 0.100.080.3, and wherein the components in the antibody drug conjugate pharmaceutical preparation ranked in descending order according to their contents are as follows: the component which has 4 small molecule toxins conjugated to the antibody (D4); the component which has 2 small molecule toxins conjugated to the antibody (D2); the component which has 6 small molecule toxins conjugated to the antibody (D6); the component which has 8 small molecule toxins conjugated to the antibody (D8); and the component which has 0 small molecule toxins conjugated to the antibody (DO).

OFFICE ACTION:
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 .
Continued Examination Under 37 CFR 1.114
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 8/21/2024 has been entered.
The rejection of claims 23-31 is withdrawn in view of Applicant’s remarks in connection with this ground of rejection.  Namely, the applied references fail to teach or suggest use of the recited reaction buffer system, the ratio of antibody to reducing agent, the ratio of antibody to toxin, the reduction reaction temperature and time, the conjugation reaction temperature and time, and the selected purification system in the preparation process.
The rejection of claims 32-35 is withdrawn in favor of the following new ground of rejection under this section:
Claim Rejections - 35 USC § 103
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.
Claims 32-35 are rejected under 35 U.S.C. 103 as being unpatentable over:
Law et al., Clin Cancer Res (2004) 10 (23): 7842–7851 (Law); and
Francisco et al., Blood. 2003 Aug 15;102(4):1458-65 (Francisco).
The applied references all describe preparation of the conjugates
Law:
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Francisco:
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The applied references may not explicitly teach the recite drug to antibody ratio (DAR) component distribution.  However, the recited conjugates were prepared in substantially the same manner as described in the specification.  In this manner, those of ordinary skill would expect that the products of the preparation would have substantially the products, including the recited DAR distribution, see MPEP 2112.01 (“Where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). “When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not.” In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).”).
In this regard, the rejected claims are not limited any particular antibody drug conjugate.
Therefore, the recited preparations are prima facie obvious.
The rejection under section 112(b) is withdrawn in favor of the following new ground of rejection under this section:
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.
Claims 32-35 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.
The criteria of ranking is not clear.  Applicant should amend the claims to clearly recite the intended criteria, e.g., amount or concentration of conjugates with the recited ratios.
Conclusion

APPLICANT REBUTTAL: REMARKS/ARGUMENTS
Claims 23-35 are currently pending in the present application. Claims 1-22 were previously canceled, without prejudice and without disclaimer. Claims 23 and 33-35 are amended herein, without prejudice and without disclaimer. No new matter is added. In view of the amendments and remarks that follow, Applicant respectfully requests reconsideration and allowance of claims 23-35.
Rej
ection of Claims Under 35 U.S.C.
§
Claims 23-35 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. In particular, the Office action states that "The claims have been amended to recite that vc-MMAE is used as a conjugation reagent. However, the formula in claim 23 also contains a maleimidocaproyl component not structurally-accounted for by vc-MMAE." See OA, page 16.
Without conceding to the propriety of the above definiteness rejection,
in an effort to ex
p
edite allowance
of the present application and to better set forth the subject matter being claimed, Applicant has amended claim 23 to recite, in the relevant part and among other things, the active steps of "(2) adding a solution of
maleimidocaproyl-vc-PAB-MMAE
in acetonitrile and water dropwise to the reaction system of step (1) and performing a coupling reaction at 4±0.5 °C for 60±10 minutes to obtain a coupling solution." The support can be found, e.g., in claim 23 itself. No new matter is added. According to the structure of the antibody drug conjugate described in claim 23 and the description of the application, it should at least now be undoubtedly determined that the reduced rituximab or a biosimilar thereof obtained in step (1) of the preparation method is to be reacted with a "linker-payload" which is maleimidocaproyl- vc-PAB-MMAE, thereby forming an antibody drug conjugate. That is, it should at least
now be clear that the reduced rituximab or a biosimilar obtained in step (1) is to be reacted with a "linker-payload," specifically maleimidocaproyl-vc-PAB-MMAE, to form an antibody-drug conjugate.
In making the rejections of claim 32-35, the Office action further states that "Claims 32-35 recite components of the instant ADC's. Some of the components are outside the recited DAR, e.g., 2, 6, 8, 0. It is unclear how these components relate to the recited ADC's. Moreover, if these are "components" of the instant ADC's, it is unclear if Applicant is claiming an ADC or a composition of different ADC's, some of which are outside the recited DAR." See OA, page 16.
Applicant respectfully traverses.
In the related art, a skilled artisan would recognize that an antibody-drug conjugate (ADC) prepared by conjugation reaction between an antibody and a toxin molecule via a linker should have a
population
in which each component has different numbers of toxin molecule coupled therein. So, a DAR value is an average. Indeed, as shown in Fig. 1 of this application, the ADC product contains component having 4 small molecule toxins conjugated to the antibody (D4), as well as other components having 2, 6, 8, or 0 small molecule toxins conjugated to the antibody (D2, D6, D8, DO), and all those different components constitute the final ADC product having an average DAR value of 4.2 ± 0.5.
In view of at least the above, Applicant respectfully submits that the amendment and the explanation should now render the rejection under 35 U.S.C. §112(b) moot and respectfully requests withdrawal of the rejections under 35 U.S.C. § 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph.
Rej
ection of Claims Under 35 U.S.C.
§
Claims 32-35 were rejected under 35 U.S.C. 103 as allegedly being unpatentable over Jain et al., Pharm Res (2015) 32:3526-3540 (Jain); in view of U.S. Publication No. 2005/ (Wahl) and Chen et al., "Determination of Drug-to-Antibody Ratio for Antibody-Drug Conjugates Purified from Serum," Agilent Technologies, pp. 1-9, 2016 (Agilent).
Claims 23-31 are rejected under 35 U.S.C. 103 as allegedly being unpatentable over Jain in view of Wahl and Agilent; in further view of Agarwal et al., Bioconjugate Chem. 2015, 26, 2, 176-192 (Agarwal); and Sun et al., Bioconjug Chem. 2005; 16: 1282-1290 (Sun); and Li Yet al., J Chromatogr A. 2015; 1393: 81-88 (Li Y); and Protein Concentration and Diafiltration by Tangential Flow Filtration, Millipore Technical Brief (Millipore), Teeters et al., Biotechnology and Bioengineering, Vol. 108, No. 6, June, 2011 (Teeters); and U.S. Publication No. 2016/0045615 (Li); and U.S. Publication No. 2017/0326251(Evans); and Package Insert Herceptin (Trastuzumab) Genetech, Inc., 2000 (Herceptin).
In order to establish a prima facie case of obviousness under 35 U.S.C. § 103, however, the Office Action must provide a clear articulated reasoning with some rational underpinning to support the legal conclusion of obviousness, demonstrating why a person having ordinary skill in the art at the time the invention was made would have combined the disclosures of the cited references to arrive at each of the claimed features of a rejected claim. MPEP 2143.03 entitled
"A
ll
Claim Limitations must be Considered"
states:
"all words in a claim must be considered in
j
udging the patentability of that claim against the prior art."
and IV; see also KSR, supra at 1740-1741. A fact
finder should be aware, of course, of the distortions caused by hindsight bias and must be cautious of arguments reliant upon ex post reasoning. One cannot use hindsight reconstruction to pick and choose among isolated disclosures of elements in the prior art to obviate the claimed invention. In re Fine, 837 F.2d 1071, 1075 (Fed. Cir. 1988).
In view of the above legal principles and tenets to the present case and to expedite allowance, Applicant respectfully submits a Declaration under 37 CFR §1.132 to further evidence the allowability of the present claimed invention and to address any Office's concerns as further traversed below.
Regarding to claims 32-35, which claim an antibody drug conjugate or an antibody drug conjugate pharmaceutical preparation comprising an antibody drug conjugate prepared according to the preparation method of claim 23, Applicant respectfully submits that both the antibody drug conjugate (claims 32-33) and the antibody drug conjugate pharmaceutical preparation (claims 34-35) claimed should not be obvious over Jain in view of Wahl and Agilent.
First, the non-obviousness of the antibody drug conjugate (ADC), which has a
population,
claimed is not only reflected by the range of DAR value (4.2±0.5) of the ADC product, but also by the proportion of each component (as recited DARO, DAR2, DAR4, DAR6, and DAR8); while as for the pharmaceutical preparation claimed, the non-obviousness of the pharmaceutical preparation is further reflected by the composition and proportion of excipients in the pharmaceutical preparation claimed.
The ADC product claimed is a mixture composed of DARO (DO), DAR2 (D2), DAR4 (D4), DAR6 (D6), DAR 8 (D8) and other components (e.g., the buffer and preparation excipients used in the claimed preparation method), with an average DAR value of 4.2 ± 0.5.
Because the pharmaceutical chemistry is a complex art, during the development of the claimed ADC product, inventors of the instant application unexpectedly discovered that increasing (the higher) the proportion of target component with DAR4 in
the ADC product is beneficial. For instance, increasing (the higher) the proportion of DAR4 component, enhances (the better) the homogeneity of ADC product, the more stable the product, and the better the pharmacokinetic and pharmacodynamics performance. The distribution of each component of the claimed ADC product is also unexpectedly beneficial.
As shown in Fig. 1 of this application, it can be clearly determined that in this application, it is evident that the target component of DAR4 (D4) in the ADC prepared is relatively high, achieving the desired goal of drug development.
It can be seen from Fig. 1 that the proportion of each component in the ADC claimed is D4 > D2 > D6 > D8 > DO from high to low; the proportion of target component D4 is the highest, and the proportion of D2 with a low DAR value is higher than that of D6 or D8 with a higher DAR value. On the one hand, because the proportion of target component D4 is the highest, the product has good homogeneity and stability; on the other hand, the proportion of the component D2 with a low DAR value is higher than that of the component D6 or D8 with a higher DAR value. The high-DAR-value
component with lower proportion (i.e., D6 or D8) can lead to a reduced off-target toxicity and a desired time required for metabolism, and help to achieve better pharmacokinetic and safety characteristics (i.e., the lower proportion of high-DAR-value components (such as D6 or D8) can reduce off-target toxicity and enhance the time required for metabolism, thereby enhancing pharmacokinetic and safety characteristics). The DO component (naked antibody) has the lowest proportion. A high proportion of DO component will affect the drug efficacy of the product, and also reflects a low conjugating efficiency. The ADC with relatively lower proportion of DO component has more advantages in drug efficacy, which also shows a higher conjugating efficiency. On the whole, the desired D4 component of the ADC of the invention account for a high proportion, and the other components account for an appropriate proportion. That is, the ADC with the lower proportion of the DO component demonstrates enhanced drug efficacy and higher conjugation efficiency, while the desired D4 component constitutes a significant proportion and the other components are present in appropriate proportions, resulting in unexpected improved pharmacokinetic and safety characteristics.
In contrast, Jain, Wahl, and Agilent should not disclosure or even suggest the features recited in currently amended claims 32-35.
In this regard, Reference 7 (Yuanyi Mang et al. submitted in response to the previous non-final action) is submitted as an illustrative counter example. Reference 7 discloses an ADC with a DAR of 3.97; however, as shown in Figure 3 in Reference 7, the component with DAR of 4 accounts for a low proportion, and components with a DAR of 0 and a DAR of 6 are the main components. That is, the reference provides an ADC with a DAR of 3.97; however, Figure 3 in Reference 7 shows that the component with a DAR of 4 is present in a low proportion, while the main components are those with DARs of 0 and 6.
A
B
C
structurc
SEC-HPLC
HIC-HPLC
M290-MC-vc-PAB-MMAE
Purity=99.0%  Average DAR=3.97
Although the average DAR value of the ADC in Reference 7 is 3.97, allegedly similar to the average DAR value of the ADC of the present invention, from the distribution of its DAR value in the figure, it can be seen that D6 and DO components account for a relatively high proportion, while the component D4 accounts for a relatively low proportion (i.e., the figure shows that the D6 and DO components are present in relatively high proportions, while the D4 component is present in a relatively low proportion).
The D6 component, a higher DAR value component with a high proportion, will be rapidly eliminated from the body because of its short half-life, thus affecting the efficacy of the ADC product; At the same time, the DO component, when accounts for a higher proportion, will lead to reduced efficacy due to no coupling of toxin. Therefore, although the average DAR value of the ADC of the present invention is close to the ADC disclosed in Reference 7, the components in the two ADCs have rather different DAR value distribution, resulting in obvious differences in their stability, efficacy and pharmacokinetics. The proportion of different DAR value components in ADC product is a key quality indicator. The reference cited by the Office does not mention the proportion of each DAR value component (such as DO, D2, D4, D6, D8) in its ADC products. The proportion of each DAR value component of the ADC of the present invention should not be obvious. Again, although the average DAR value of the ADC in the present invention is close to that in Reference 7, the differing proportions of DAR components (DO, D2, D4, D6, D8) result in significant differences in stability, efficacy,
and pharmacokinetics, making the proportion of each DAR value component a key critical quality indicator that should not be obvious from the reference cited by the Office.
Second, Applicant would like to point out that the ADC or the pharmaceutical preparation claimed is prepared according to the preparation method of Claim 23. According to the preparation method of Claim 23, clearly the ADC product and the pharmaceutical preparation obtained includes the anti-CD20 antibody drug conjugate product, as well as preparation excipients which are histidine hydrochloride, trehalose and polysorbate 80, with a pH of 6.5; more specifically, preparation excipients are derived from the HT preparation buffer recited in claim 23 and as described in paragraph [0106] of the description which contains 3.18 g of L-histidine, 70 g of trehalose dihydrate, and 0.2 mL of Tween 80 per L, with pH adjusted to 6.5 using 0.5 mol/L hydrochloric acid.
The skilled artisan may know that the buffer system and pH value thereof are crucial to the stability of a pharmaceutical preparation, but there is no obvious guideline on how to choose the buffer system. As disclosed in the Declaration, the inventors' insight is to test two buffer systems to establish a suitable buffer for the claimed ADC product: sodium citrate buffer (set at pH 6.5) and histidine hydrochloride buffer (set at pH values of 6.1, 6.3, 6.5, 6.7, and 6.9). For details, see Table 10-4 of the Declaration, provided below. .
In the above buffer systems, the protein concentration of the ADC product was
diluted into 10 mg/ml, and the preparations obtained were stored at -80 0C, 4 0C, or 37
C respectively, and then at day 0 and after storage of 1 week, 3 weeks, or 4 weeks, the
appearance, protein concentration, and purity via SEC and HIC of the preparations
were measured; and DSC (Differential Scanning Calorimetry) were performed at day 0.
See Table 10-5, Table 10-6, Figure 10-2, Figure 10-3, and Figure 10-4 of the
Declaration for the experimental results, which are provided as follows too.
Blue: before dialysis and af er- the nixing; PinAc after dialysis;Red: sample 0; Biyn: sample
1; Light blue: sample 2; Light green: sample 3; Grey: sample 4; Green: sample 5
Figure 10-2. 0-day SEC-HPIC diagram
ofthe samples with different pH values
Figu 10-3 Typical SEC-HPLC diagram of the sampIes wtit diferent pH values (samples 0, 1, and 2 after4 weeks of stomge) frstabilityinvestigation
Flure 10-4 TypicaI SEC-EPIC daam ofthe aiples with different ph
va
lues
(samples 3, 4, and 5 after 4 weeks of storap) for stability investigation
The results unexpectedly showed that:
1) With respect to the stability of the preparation samples with different pH
values:
It can be seen that a large number of white fine solid precipitates emerged in Sample 0 (pH 6.5) after Sample 0 was stored at 37 0C for 1 week; Sample 1 (pH 6.5), Sample 4 (pH 6.7) and Sample 5 (pH 6.9) showed slight whitening after they were stored at 37 0C for 4 weeks, while their appearance did not change after they were stored at -80 0Cand 4 0C for 4 weeks; the appearance of Sample 2 (pH 6.3) and Sample
3 (pH 6.5) did not change after they were stored at -80 0C, 4 0Cand 37 0C for 4 weeks.
The above results showed that Sample 0 (pH 6.5) using sodium citrate buffer system was unstable, and Samples 1-5 using histidine hydrochloride buffer system was stable, especially Sample 2 and Sample 3 with pH 6.3 and pH 6.5, respectively.
2)With respect to the protein content:
It can be seen that the protein content of Sample 0 (pH 6.5) with sodium citrate buffer system decreased significantly after Sample 0 was stored at 37 0C for 1 week, and further decreased during the third week. The protein content of Samples 1-5 with histidine hydrochloride buffer system did not change significantly after the samples were stored at -80 C, 4 0C and 37 0C for 4 weeks.
3)With respect to the SEC-HPLC purity:
SEC purity showed that there was no difference between the samples with different pH values at day 0(see Figure 10-2). All the samples stored at 37 0C for 4 weeks exhibited aggregated, and the degree of aggregation at acceleration of 4 weeks was: Sample 0 (pH 6.5) > Sample 1 (pH 6.1), Sample 2 ,(pH 6.3), Sample 4 (pH 6.7), Sample 5 (pH 6.9) > Sample 3 (pH 6.5); see the green circle in Figure 10-3 and Figure 10-4.
The above results showed that Sample 3 using histidine hydrochloride as buffer system and having pH 6.5 unexpectedly has the best stability.
Therefore, histidine hydrochloride is the critical preparation buffer for preparing a pharmaceutical preparation of the anti-CD20 antibody drug conjugate of the invention, and the pH value should critically be set to 6.5.
To sum up, the proportion of each component in the ADC product claimed is D4>D2>D6>D8>DO from high to low, which makes the ADC product has excellent pharmacodynamics, pharmacokinetics and safety characteristics. At the same time, in addition to the ADC product, pharmaceutical excipients are contained in the pharmaceutical preparation because the ADC product is prepared according to the preparation method of claim 23. The specific critical claimed buffer system and the pH value enable the pharmaceutical preparation as claimed to have desired stability. Applicant respectfully submits that the references cited by the Office should not disclose or even suggest these features, and those features should not be obvious over the references of record.
For at least the reasons above, Applicant respectfully submits that claims 32-35 of the present application should be patentably distinguished from the references of record and thus respectfully request that the rejections of claims 32-35 over Jain, Wahl, and Agilent be withdrawn.
Regarding claims 23-31, Applicant respectfully submits the preparation method claimed should not be obvious over Jain in view of Wahl and Agilent, in further view of Agarwal, Sun, Li Y, Millipore, Teeters, Li, Evans, and Herceptin.
Applicant notes that the Office cites references of record and deems that, those references have disclosed antibody reduction, conjugation reaction, reaction termination and purification steps, etc. adopted for preparing ADCs, and although the references did
not disclose parameters such as feeding ratios of reaction reagents, reaction temperature, time and other parameters defined in the claims, but they are results- effective parameters because they affect the efficiency of the process, so their optimization is well within the ordinary skill of the purview of those of ordinary skill, and therefore, prima facie obvious.
Applicant respectfully disagrees. Although the process of preparing ADCs through conjugating via cysteines usually includes antibody reduction, conjugation reaction, termination reaction and purification steps, the selection of parameters used in these reaction steps is critical. Those skilled in the art may be motivated to optimize the parameters, but the preparation process involves multiple key parameters (such as reaction buffer, reductant to antibody feeding ratio, antibody to toxin feeding ratio, reduction reaction temperature and time, conjugation reaction temperature and time, etc.), and these key parameters interact with each other, resulting in a high unpredictability, thereby affecting multiple characteristics of the ADC product obtained(such as the average DAR value, proportion of components with different DAR values, contents of aggregates and impurities, stability, efficacy and pharmacokinetics, etc.), which brings great difficulties to find a suitable preparation process and incurs considerable cost. That is, it should be apparent to one skilled in the art that these variables interact in complex ways, leading to high unpredictability and affecting multiple characteristics of the resulting ADC product (such as average DAR value, proportion of components with different DAR values, aggregate and impurity content, stability, efficacy, and pharmacokinetics). This complexity makes finding a suitable preparation process extremely challenging and costly.
In this case, during the investigations on the preparation process, inventors of the present application unexpectedly discover that when 0.05 M phosphate buffer (pH 7.6) was used as the reaction buffer, the feeding ratio of antibody to reducing agent was
about 1:3, the feeding ratio of antibody to toxin was about 1:7.5, the reduction reaction was performed at about 25 0C for about 90 minutes, and the coupling reaction was performed at about 4 0C for about 1 hour, the ADC product prepared had excellent batch-to-batch consistency and excellent distribution of different DAR components, and the desired component(the component with DAR4) is highly enriched.
(1) Regarding the selection of reaction buffer:
Please refer to the Declaration, 0.2 M Tris HCI buffer (pH 8.0), 0.2 M HEPES buffer (pH 7.5), 0.2 M phosphate buffer (pH 7.6), and 0.05 M phosphate buffer (pH 7.6) were screened. The results showed that, the reaction mixture was clear after adding the toxin when 0.05 M phosphate buffer (pH 7.6) was used as the reaction buffer, and no precipitates appeared after low-temperature storage. When other buffers were used as the reaction buffer respectively, flocculent precipitates appeared in the reaction mixture after adding the toxin, or precipitates appeared in the reaction mixture after low- temperature storage.
(2) Regarding the conjugation reaction temperature:
Free sulfhydryl groups generated after the reduction of antibody disulfide bond react with the maleimide linker group of the added toxin molecule. That reaction belongs to Michael addition reaction, and the theoretical reaction temperature is 0 C. However, in the present preparation method claimed, the reaction temperature can be controlled at about 4 0C through a temperature controlled circulating water bath system and ethylene glycol used as the refrigerant, so that the temperature control can be more accurate and convenient, thereby improving the reaction efficiency. In other words, the free sulfhydryl groups generated after the reduction of the antibody disulfide bond react with the maleimide linker group of the added toxin molecule in a Michael addition reaction, which theoretically occurs at
C.
However, in the claimed preparation method, the reaction temperature is unexpectedly controlled at about
using a temperature-controlled circulating water bath system with ethylene glycol as the refrigerant. This allows for more accurate and convenient temperature control, thereby improving reaction efficiency.
(3) Regarding the reducing agent and the purification step:
Wahl taught using DTT as the reducing agent, and after the reduction reaction, G25 column is used and the column is eluted with PBS to change the liquid; and in that prior art, after the conjugation reaction and termination reaction, the reaction mixture was passed through the G25 column again and eluted with PBS to change the liquid. Clearly, the process taught by Wahl adopts two-step purification, which increases the number of operation steps and reduces the yield at the same time.
In contrast, in the preparation process as claimed, TCEP is insightfully selected by the inventors and used as the reducing agent and TFF system is used for purification accordingly. TCEP is a highly efficient disulfide reducing agent, and can exert its reduction effect in a wide range of pH values. Further, TCEP will not affect the
conjugation reaction between antibody and toxin-linker, therefore TCEP does not need to be removed after reduction reaction, so only one-step purification with TFF system is required, and so the process is simplified and the yield will be improved. Although TFF system may be a conventional choice in the art, one-step purification using TFF system and TECP, instead of the two-step purification as taught by Wahl, is not obvious for those skilled in the art.
(4) Regarding the feeding ratio of antibody to reductant and the feeding ratio of antibody to toxin:
The results of screening tests in this regard conducted by Applicant showed that, if the feeding ratio of antibody to reducing agent was higher than 1:3 which is defined in the invention, for example, 1:2.5, and the feeding ratios of antibody to toxin were 1:4, 1:7, and 1:10, the average DAR values of ADC products were 2.34, 3.99, and 3.99, respectively. The distribution of components having different DAR values is shown in Figure 9-45 of the Declaration, which is provided as follows.
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It can be seen from Figure 9-45 that when the feeding ratio of antibody to reductant agent was 1:2.5, and the feeding ratio of antibody to toxin was 1:7 or 1:10, the average DAR of the ADC product obtained was 3.99, but the proportion distribution of components with different DAR values was very unsatisfactory. The proportion of D2 component was too high, but the proportion of the desired DAR4 components was relatively lowered, and the homogeneity was decreased, which would have a negative impact on the efficacy of the ADC product.
Further, if the feeding ratio of antibody to reducing agent was 1:3(as defined and claimed currently)and the feeding ratios of antibody to toxin were 1:5, 1:6, and 1:8, the average DAR values of ADC products were 2.59, 3.88 and 4.52. The distribution of components having different DAR values is shown in Figure 9-46 of the Declaration, which is provided as follows.
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It also can be seen from Figure 9-46 that when the feeding ratio of antibody to reductant agent was 1:3 and the feeding ratio of antibody to toxin is 1:6, the average DAR of the ADC product obtained was 3.88, close to 4. However, in the ADC product, the proportion distribution of components with different DAR values was not satisfactory (very unsatisfactory). The proportion of the desired component with DAR4 was relatively low, while the proportion of component with DARO, DAR 2, DAR 6, or DAR 8 is relatively high, which will seriously affect efficacy, pharmacokinetics and other characteristics of the ADC product. When the ratio of antibody to toxin was 1:8, the proportion distribution of components with different DAR values was appropriate, the product homogeneity was good, the efficacy and pharmacokinetics were excellent.
Further, in order to make the average DAR of ADC product closer to the target value of 4.2, the feeding ratio of antibody to toxin was further adjusted to 1:7.5. With this feeding ratio, three batches (C16205-20160106001, 016205-20160106002, 016205- 20160106003) of ADC products were produced. The average DAR values of ADC products of the batches were 4.22, 4.35 and 4.41, respectively. The proportion distribution of components with different DAR values of the batches is shown in Figure 9-47 of the Declaration, which is provided as follows.
o
The HIC chromatogram of three batches of ADC products were combined and provided in Figure 5 of the Applicant's previous patent CN108452318B (previously submitted through IDS), which is provided as follows.
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DAR value of the ADC product obtained could be well controlled within the range of 4.2 ± 0.5 or even 4.2 ± 0.3, and further, the proportion distribution of components with different DAR values was appropriate also, for example, the proportion of desired component with DAR4 was the highest, while the proportions of components with DO, D2, D6 and D8 were relatively low and in an appropriate order. These characteristics result in an ADO product with excellent homogeneity, pharmacokinetics, and pharmacodynamics. In contrast, other feeding ratios of antibody to reducing agent and antibody to toxin (such as 1:2.5 and 1:7; 1:2.5 and 1:10; 1:3 and 1:6) may yield an average DAR value close to 4, but the distribution of components with different DAR values is unsatisfactory, negatively impacting the homogeneity, pharmacokinetics, and efficacy of the ADC products (i.e., although other feeding ratios of antibody to reducing
agent and feeding ratios of antibody to toxin (such as antibody: reducing agent=1:2.5 and antibody: toxin =1:7; antibody: reducing agent=1:2.5 and antibody: toxin =1:10; antibody: reducing agent=1:3 and antibody: toxin =1:6)lead to an average DAR value close to 4, the proportion distribution of components with different DAR values in the ADC product obtained is dissatisfactory, which adversely affects the homogeneity, pharmacokinetics and efficacy of ADC products). Therefore, the feeding ratio of antibody to reducing agent of 1:3 in combination with the feeding ratio of antibody to toxin of 1:7.5 should not be obvious.
The detection results of the three batches of products as shown in the above Figure 5 prove that, the ADC product prepared by the preparation process claimed has good batch-to-batch consistency, and its average DAR value can be well controlled within the range of 4.2 ± 0.5 or even 4.2 ± 0.3. Here, the preparation process claimed has good reproducibility and stability, which is beneficial to product quality control and large-scale commercial production.
To sum up, the selected reaction buffer system and reducing agent, the ratio of antibody to reducing agent, the ratio of antibody to toxin, the reduction reaction temperature and time, the conjugation reaction temperature and time, and the selected purification system in the preparation process claimed are obtained by the applicant through overall design. The parameters are not in isolation, but are interrelated. For example, the ratio of antibody to reducing agent and the ratio of antibody to toxin are interrelated, and the selected reducing agent and the selected purification system are interrelated, which cannot be obtained by simple parameter optimization. As mentioned above, the preparation process claimed as a whole can achieve excellent technical effects. On the one hand, the preparation process itself is easy to operate and can be accurately controlled, with good repeatability, and is suitable for large-scale commercial production. On the other hand, the target component with DAR4 in the ADC product prepared by the preparation process is highly enriched, and the proportion distribution
of components with other DAR values is appropriate, so that the ADC product has high homogeneity and excellent pharmacokinetics, efficacy and stability. In other words, the preparation process claimed involves a carefully designed combination of reaction buffer system, reducing agent, antibody-to-reducing agent ratio, antibody-to-toxin ratio, reduction reaction temperature and time, conjugation reaction temperature and time, and purification system. These parameters are interrelated and cannot be merely optimized in isolation. For instance, the ratios of antibody to reducing agent and antibody to toxin, as well as the chosen reducing agent and purification system, are interconnected. This holistic approach results in a process that is easy to operate, accurately controlled, repeatable, and suitable for large-scale commercial production. Additionally, the process yields an ADC product with a high enrichment of the DAR4 component and an appropriate distribution of other DAR components, leading to high homogeneity, excellent pharmacokinetics, efficacy, and stability.
In addition, the preparation process claimed has a definitive role on the ADC product itself. The ADC product prepared by the preparation process not only has the average DAR value in the range of 4.2 ± 0.5, but also has a high enrichment of desired component with DAR4, and the proportion of components with different DAR values from high to low is D4>D2>D6>D8>D0.
The screening experiments on different parameters, especially the ratios of antibody to reducing agent, and the ratios of antibody to toxin (as shown above)showed that, different parameters will lead to huge changes in the proportions of ADC components. The reference documents cited by the Office do not disclose the distribution of ADC components with different DAR values, nor it can be determined that the ADC product prepared by a preparation process in the reference documents has the same or similar distribution of ADC components with different DAR values as the ADC product obtained according to the preparation method claimed. Based on the above, the ADC preparation process of the invention should not be obvious. The preparation
process has a definitive role on the ADC product other than the DAR value. Therefore, the ADC product of the invention should also not be obvious, either.
Under MPEP Section 2144.05 (III), prima facie obviousness may be rebutted by showing the criticality of the claimed range. As discussed above, Applicant has set forth the criticality of the selected reaction buffer system and reducing agent, the ratio of antibody to reducing agent, the ratio of antibody to toxin, and the selected purification system, and thus those discussed criticalities should provide further evidence that claims 23-35 should be patentably distinguished from and non-obvious over the references of record.
Indeed, the recited biological drug is very complex, especially ADC products. Due to the large molecular weight and complex spatial structure of biomolecules, a change in any parameter may result in high uncertainty and unpredictability, as compared to chemical drugs or conventional mechanical structures. So, Applicant respectfully requests the inventiveness of this application based on the understanding that biopharmaceutical development is very complex and high uncertainty.
For at least the reasons detailed above, claims 23-35, as a whole, should at least now be patentably distinguishable over the references of record. Thus, Applicant respectfully requests the Office that the rejection under 35 U.S.C. § 103 be withdrawn, and that claims 23-35 be allowed.
In addition, claims 33 and 35 are amended to correct clerical informality, claim 34 is amended for clarity. No new matter is added.
Concluding Remarks
In view of the foregoing amendments and remarks, Applicant earnestly solicits a timely issuance of a Notice of Allowance. If there are any remaining issues that can be
addressed over the telephone, the Examiner is cordially invited to call Applicant's attorney at the number listed below.
Payment of any fee required to enter and process this amendment is provided. The Director is authorized to charge any underpayment of fees or credit any overpayment of fees to Deposit Account No. 03-1728.
Respectfully submitted, LEWIS ROCA ROTHGERBER CHRISTIE LLP

NEXT OFFICE ACTION: 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 .
The following is an examiner’s statement of reasons for allowance:
The rejection under section 103 is moot since the rejected claims have been canceled.
The rejection under section 112(b) is moot since the rejected claims have been canceled.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion