**10. Antibody-drug conjugation**

*Advances in Precision Medicine Oncology*

non-small cell lung cancer [48].

**Figure 7.**

*(arrowed).*

**9. Tumor cell lysis in vitro**

lacking the target antigen.

internal organs of liver, spleen, stomach, intestine, colon, and pancreas were histologically examined, and no visible pathologic changes were identified. The sparing of organs in anti-AGR2 tumor targeting was also reported by another group [46]. The mechanism behind this epitope-dependent phenomenon is unknown but could be related to a reported observation of increased tumor inhibition by an antibody to AGR3 in combination with the chemodrug cisplatin [47]. AGR3 is a close family member of AGR2. Both AGR2 and AGR3 tend to be elevated in cancer, though to different levels as found in prostate cancer [10]. The combination of monoclonal antibody plus biological inhibitors are being pursued to treat more successfully

*Drug inhibition of tumor growth enhancement by P1G4. (A) Representative immunohistochemistry images show the effect (from top to bottom) of IgG control, P1G4 (P1) alone, Gemcitabine (GEM) alone, P1 + GEM. Ki67 staining indicates that tumors treated with drug still had high proliferation rate, which was limited in P1 + GEM tumors. CD3 shows T cell infiltration in the GEM and P1 + GEM groups. (B) Tumors resected at week 6 from the different treatment groups are compared. The smallest size is found in the P1 + GEM group* 

In our earlier work, 51Cr radiolabeled target cancer cells were exposed to TAA antibodies and human serum (as a source of complement factors) or peripheral blood leukocytes [43, 49]. By CDC, the chimeric antibody generated higher cytotoxicity at all complement dilutions. By ADCC, at a ratio of 100:1 blood leukocytes to target cells, the chimeric lysed a greater fraction of the cancer cells and gave 50% cytolysis at 100-fold lower concentration than the mouse antibody. ADCC was observed at a 3:1 ratio of effector to target cells when the chimeric (at 2.5 μg/ml) was used. Cell killing was specific because ADCC was not observed with cell lines

To test the anti-tumor effect of chimeric antibodies, we incubated PC3 prostate cancer cell line in the presence of donated human serum. Like pancreatic cancer cells, cell surface expression of eAGR2 was found on PC3 cells. Spin-concentrated chimeric IgG was used with human serum for CDC. PC3 cells were incubated with freshly donated human serum and added AGR2 antibodies. There was no observable effect on cell viability in the culture well with human serum only, as was the well with mouse P3A5 + serum. In the well with a cocktail of chimeric IgG1, IgG2, and IgG4, cell growth was inhibited with well surface showing areas devoid of cells,

**104**

In addition to antibodies that rely on immune system components, a cytotoxic drug can be linked directly to the AGR2 antibodies to produce an antibody-drug conjugate (ADC). ADC delivers the drug payload to the target organs or tissues [52]. For anti-AGR2 ADC, drug compounds were constructed by covalently linking poly(N-isopropylacrylamide) to both P1G4 and P3A5 via carbodiimide chemistry. The linking polymer was synthesized by reversible addition fragmentation chain transfer (RAFT) with a carboxylate chain end [53]. To conjugate antibodies, the carboxylate was converted to an active ester for formation of an amide bond to lysine residues [53–56]. We have also developed a block copolymer with tetrafluorophenyl (TFP) ester monomers to drive the antibody conjugation [57]. The resulting anti-AGR2 ADC were confirmed by gel electrophoresis, which showed the larger molecular weight products compared to the unconjugated antibodies (**Figure 8**). After the size-exclusion chromatography, the purified ADC were shown by ELISA to bind AGR2 (**Figure 8**). Sample solutions containing a constant AGR2 concentration were mixed with ADC from 125 to 1,000 ng/ml. ELISA measured the unbound AGR2 with higher ADC concentrations resulting in less free AGR2 in the solution. Thus, our conjugation chemistry did not affect appreciably the antigen binding affinity of the antibodies. Polymer chains with improved loading capacity by incorporating functional groups [58] could be developed for conjugation to docetaxel, doxorubicin, and other drugs. To improve delivery efficiency, the polymer can be engineered to increase circulation time, and the polymer composition can be modulated.

#### **Figure 8.**

*Antibody drug conjugate. The left panel shows the conjugated products (lane 1) vs. unconjugated antibody (lane 4). The right plot shows more AGR2 bound (percentage, y-axis) with higher ADC concentrations of the conjugate (in ng/ml, x-axis).*
