**12. Future work and developments**

#### **12.1 Preclinical testing of chimeric antibodies**

With the availability of chimeric antibodies and ADC, we can carry out animal studies to assess safety and efficacy of anti-AGR2 therapy, especially in PDX models available in prostate and bladder cancers. The LuCaP lines recapitulate the molecular heterogeneity of metastatic castration-resistant prostate cancer. Overall, a majority of molecular events characterized in human prostate tumors are found in LuCaP such as *AR* amplification, *PTEN* loss, *RB1* deletion, DNA damage response deficiencies [40, 61]. These LuCaP lines can be selected for study: AGR2+ LuCaP 23.1, 35, 136, 147 and AGR2<sup>−</sup> LuCaP 145.1 to test P1G4-ADC, P3A5-ADC, P1G4 + docetaxel, P3A5 + docetaxel. With regard to AGR2 expression, LuCaP 23.1 and LuCaP 147 have relatively high levels, while small cell carcinoma LuCaP 145.1 as control has no expression [27]. Complete effect from anti-AGR2 would be expected for the former examples and no effect for the latter. For IgG control, we can transfect 293F cells with a construct containing H chain of P3A5 and L chain of P1G4 or H chain of P1G4 and L chain of P3A5, which would be expected to produce an AGR2 non-binding V domain. Green fluorescence protein (GFP)-labeled chimeric antibodies could be used for tumor localization (to show no labeling of iAgr2+ normal organs) in place of radioisotopes, if necessary, to add to our pancreatic cancer PDX result. The study will also test if P1G4 has an enhancement effect in tumor growth inhibition by docetaxel as shown by this antibody in the Gemcitabine study. Response by LuCaP lines to docetaxel treatment varied substantially [40]. A high dose (20 mg/kg) generally produced growth suppression and survival benefits.

**107**

*Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2*

The optimal antibody concentration (μg/ml) for 106

ADC is currently under large clinical trials [64, 67].

AGR2+

be effective against PSMA<sup>−</sup>

Only LuCaP 86.2 showed response at a low dose. Others like LuCaP 35 showed reduced survival as monitored by body weight loss despite tumor growth inhibition. We will test whether the response to docetaxel will be less variable and more pronounced with the addition of P1G4 (but not P3A5). The P1G4-docetaxel ADC may act in an equivalent way as P1G4 + docetaxel. Docetaxel was the first chemotherapeutic drug shown to prolong patient survival, and is in widespread use [62]. The different anti-AGR2-ADC are tested through intraperitoneal injection twice a week for four weeks. In our pancreatic cancer study, a 5 mg/kg antibody concentration was shown to be effective in the P1G4 + Gemcitabine arm. LuCaP tumor volume and body weight are monitored, and treatment will last four weeks. Tumors are collected at study end for histology, and the internal organs are examined for anti-Agr2 effect, if any. Serum is collected for AGR2 measurements by ELISA. Serum PSA may also be measured to see if there is concordance between these two biomarkers. Other parameters to try include higher antibody concentration, longer treatment time. The in vitro adapted LuCaP cells will allow us to determine the effective dose of serum and effector cell donations from multiple individuals (and if available, blood from cancer patients whose immune response may be compromised by their disease) in CDC and ADCC, respectively, mediated by the chimeric IgG.

is possible that a higher dose is required since AGR2 is secreted by the tumor cells where a portion of the antibodies might be bound to the free antigen. This in vitro assay is in effect an immune system model for the human body. PDX lines can also be grown as spheroids/organoids in vitro [63]. Organoids could be used to represent a solid tumor mass *vs*. monolayer culture to test the efficacy of chimeric antibodies. In the past years, an ADC to PSMA was shown to produce clinically relevant decline in serum PSA and circulating tumor cell counts in metastatic castration resistant, taxane-experienced and chemo-naived prostate cancer patients [64]. At a working dose of 2.3 mg/kg, side effects reported in some patients include neutropenia, fatigue, electrolyte imbalance, anemia and neuropathy. A small number died from disease progression. PSMA is a membrane metalloenzyme (FOLH1) found in the kidney, small intestine, central and peripheral nervous systems [65]. Non-exclusivity of this TAA to prostate cancer likely accounts for the range of side effects observed. The co-targeting of normal cells has led to recall of, for example, gemtuzumab because of severe complications since the TAA, CD30, is present on both leukemic blast and normal cells [66]. Importantly, not all prostate cancer cells express PSMA. Anti-PSMA-MMAE (monomethyl auristatin E) was shown less or not effective against tumors with low or null PSMA expression [59]. In this efficacy study using LuCaP cells, complete tumor regression for LuCaP 96CR with the highest PSMA expression was found, and no response for LuCaP 58 with the lowest PSMA expression [59]. However, intermediate effect was found for LuCaP 77 and LuCaP 105, two lines with similar PSMA expression levels as LuCaP 96CR. Why the difference was not explained. Thus, the inherent pitfall in prescribing anti-PSMA therapy by itself is selection of PSMA-negative tumors. Nonetheless, this PSMA

The rationale of our developing a second prostate cancer immunoreagent is that anti-eAGR2 could complement anti-PSMA when used in combination, which could

Note LuCaP 35 is negative for PSMA. This particular model is used to show whether anti-AGR2 will inhibit its growth where anti-PSMA could not. Two other attributes additionally bolster our rationale. eAGR2, unlike PSMA, is tumor-specific as normal cells do not express eAGR2, and metastatic prostate cancer cells express high levels of eAGR2. The two AGR2 epitopes (P1G4 and P3A5) targeted could also ensure that allelic changes in one of the epitopes will not affect susceptibility of the

AGR2−

, and PSMA+

AGR2+

cancer cells.

, PSMA+

cancer cells is determined. It

*DOI: http://dx.doi.org/10.5772/intechopen.96492*

#### *Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2 DOI: http://dx.doi.org/10.5772/intechopen.96492*

*Advances in Precision Medicine Oncology*

negative or low for AGR2.

**12. Future work and developments**

LuCaP 23.1, 35, 136, 147 and AGR2<sup>−</sup>

**12.1 Preclinical testing of chimeric antibodies**

**11. Adaptation of PDX LuCaP lines to in vitro culture**

For therapeutic testing, the LuCaP series of >40 different models established from patient tumor samples donated at autopsies and surgeries provide an invaluable resource than long-used cultured cell lines. They have been molecularly and pharmacologically characterized, and encompass a large spectrum of the disease course and representative of human prostate cancer [39]. Transcriptomics and genomics data have shown that the gene expression of these cancer cells was concordant with that of the human tumors from which they originated. Indeed, these models were used in the preclinical study to determine efficacy of anti-prostatespecific membrane antigen (PSMA) ADC as they show a range of PSMA expression [59]. For AGR2, concordant expression has been determined by DNA microarrays [40], immunostaining [27], and ELISA measurement of secreted AGR2 [20, 38]. Similar to prostate cancer patient specimens, the adenocarcinoma lines are positive for AGR2 while the small cell carcinoma including some non-adenocarcinoma are

Their utility could be increased if they can be grown outside the mouse for in vitro testing of ADCC and CDC. We showed that LuCaP cells prepared from freshly excised tumors could be successfully cultured long-term in the presence of irradiated mouse embryonic fibroblasts (MEF) as feeder [60]. Furthermore, LuCaP cells could be viably frozen using a protocol for stem cells [60], which makes constant harvests from animals unnecessary. Both adenocarcinoma and small cell carcinoma LuCaP lines could be thus grown in culture (unpublished data). The in vitroadapted cell lines with differential AGR2 expression could be used to determine the molecular mechanism controlling AGR2 expression in cancer cells. The same methodology can be used to adapt CoCaB cells for in vitro growth and testing.

With the availability of chimeric antibodies and ADC, we can carry out animal studies to assess safety and efficacy of anti-AGR2 therapy, especially in PDX models available in prostate and bladder cancers. The LuCaP lines recapitulate the molecular heterogeneity of metastatic castration-resistant prostate cancer. Overall, a majority of molecular events characterized in human prostate tumors are found in LuCaP such as *AR* amplification, *PTEN* loss, *RB1* deletion, DNA damage response deficiencies [40, 61]. These LuCaP lines can be selected for study: AGR2+

P1G4 + docetaxel, P3A5 + docetaxel. With regard to AGR2 expression, LuCaP 23.1 and LuCaP 147 have relatively high levels, while small cell carcinoma LuCaP 145.1 as control has no expression [27]. Complete effect from anti-AGR2 would be expected for the former examples and no effect for the latter. For IgG control, we can transfect 293F cells with a construct containing H chain of P3A5 and L chain of P1G4 or H chain of P1G4 and L chain of P3A5, which would be expected to produce an AGR2 non-binding V domain. Green fluorescence protein (GFP)-labeled chimeric antibodies could be used for tumor localization (to show no labeling of iAgr2+ normal organs) in place of radioisotopes, if necessary, to add to our pancreatic cancer PDX result. The study will also test if P1G4 has an enhancement effect in tumor growth inhibition by docetaxel as shown by this antibody in the Gemcitabine study. Response by LuCaP lines to docetaxel treatment varied substantially [40]. A high dose (20 mg/kg) generally produced growth suppression and survival benefits.

LuCaP 145.1 to test P1G4-ADC, P3A5-ADC,

**106**

Only LuCaP 86.2 showed response at a low dose. Others like LuCaP 35 showed reduced survival as monitored by body weight loss despite tumor growth inhibition. We will test whether the response to docetaxel will be less variable and more pronounced with the addition of P1G4 (but not P3A5). The P1G4-docetaxel ADC may act in an equivalent way as P1G4 + docetaxel. Docetaxel was the first chemotherapeutic drug shown to prolong patient survival, and is in widespread use [62].

The different anti-AGR2-ADC are tested through intraperitoneal injection twice a week for four weeks. In our pancreatic cancer study, a 5 mg/kg antibody concentration was shown to be effective in the P1G4 + Gemcitabine arm. LuCaP tumor volume and body weight are monitored, and treatment will last four weeks. Tumors are collected at study end for histology, and the internal organs are examined for anti-Agr2 effect, if any. Serum is collected for AGR2 measurements by ELISA. Serum PSA may also be measured to see if there is concordance between these two biomarkers. Other parameters to try include higher antibody concentration, longer treatment time. The in vitro adapted LuCaP cells will allow us to determine the effective dose of serum and effector cell donations from multiple individuals (and if available, blood from cancer patients whose immune response may be compromised by their disease) in CDC and ADCC, respectively, mediated by the chimeric IgG. The optimal antibody concentration (μg/ml) for 106 cancer cells is determined. It is possible that a higher dose is required since AGR2 is secreted by the tumor cells where a portion of the antibodies might be bound to the free antigen. This in vitro assay is in effect an immune system model for the human body. PDX lines can also be grown as spheroids/organoids in vitro [63]. Organoids could be used to represent a solid tumor mass *vs*. monolayer culture to test the efficacy of chimeric antibodies.

In the past years, an ADC to PSMA was shown to produce clinically relevant decline in serum PSA and circulating tumor cell counts in metastatic castration resistant, taxane-experienced and chemo-naived prostate cancer patients [64]. At a working dose of 2.3 mg/kg, side effects reported in some patients include neutropenia, fatigue, electrolyte imbalance, anemia and neuropathy. A small number died from disease progression. PSMA is a membrane metalloenzyme (FOLH1) found in the kidney, small intestine, central and peripheral nervous systems [65]. Non-exclusivity of this TAA to prostate cancer likely accounts for the range of side effects observed. The co-targeting of normal cells has led to recall of, for example, gemtuzumab because of severe complications since the TAA, CD30, is present on both leukemic blast and normal cells [66]. Importantly, not all prostate cancer cells express PSMA. Anti-PSMA-MMAE (monomethyl auristatin E) was shown less or not effective against tumors with low or null PSMA expression [59]. In this efficacy study using LuCaP cells, complete tumor regression for LuCaP 96CR with the highest PSMA expression was found, and no response for LuCaP 58 with the lowest PSMA expression [59]. However, intermediate effect was found for LuCaP 77 and LuCaP 105, two lines with similar PSMA expression levels as LuCaP 96CR. Why the difference was not explained. Thus, the inherent pitfall in prescribing anti-PSMA therapy by itself is selection of PSMA-negative tumors. Nonetheless, this PSMA ADC is currently under large clinical trials [64, 67].

The rationale of our developing a second prostate cancer immunoreagent is that anti-eAGR2 could complement anti-PSMA when used in combination, which could be effective against PSMA<sup>−</sup> AGR2+ , PSMA+ AGR2− , and PSMA+ AGR2+ cancer cells. Note LuCaP 35 is negative for PSMA. This particular model is used to show whether anti-AGR2 will inhibit its growth where anti-PSMA could not. Two other attributes additionally bolster our rationale. eAGR2, unlike PSMA, is tumor-specific as normal cells do not express eAGR2, and metastatic prostate cancer cells express high levels of eAGR2. The two AGR2 epitopes (P1G4 and P3A5) targeted could also ensure that allelic changes in one of the epitopes will not affect susceptibility of the

cancer to anti-AGR2. For bladder cancer, we will carry out a similar study employing the AGR2+ CoCaB lines to test the ADC and P1G4 + cisplatin. Cisplatin is more commonly used to treat this cancer, sometimes in combination with Gemcitabine [68]. In vitro-adapted CoCaB lines are similarly used for CDC and ADCC testing.

Cancer treatment by antibody is well established and proven to be effective. One good example is trastuzumab to target HER2/EGFR (CD340) for a subset of breast cancer and non-small cell lung cancer [69, 70]. The major obstacle to more success in antibody therapy is the time-consuming need to discover TAA for each type of cancer that at a minimum is expressed by only a few normal cell types. eAGR2 is not only expressed in prostate cancer cells but also multiple tumor types so that anti-AGR2 would have a much wider application than just treating one or two cancers. Our experiments will demonstrate the validity of this claim.

#### **12.2 Lineage relationship between AGR2+ and AGR2− prostate cancer**

The use of anti-AGR2 might lead to the selection of AGR2lo/− non-adenocarcinoma cancer including small cell carcinoma. The introduction of newer antiandrogen therapies of late has led to an almost 20% patients presenting small cell carcinoma at treatment failure [71]. Of critical importance is finding a means to prevent the emergence of AGR2<sup>−</sup> tumors.

**Figure 9** shows our model of prostate cancer differentiation relating luminallike AGR2+ adenocarcinoma to AGR2− more stem-like small cell carcinoma [72]. Stem-like cancer cells could arise from de-differentiation of luminal-like cancer cells. This process is akin to reprogramming of somatic cells via forced expression of a set of stem cell transcription factors (scTF) to induced pluripotent stem (iPS) cells [73]. We demonstrated that prostate adenocarcinoma cells could be so reprogrammed to stem-like, small cell carcinoma-like derivatives with scTF LIN28A, NANOG, POU5F1, SOX2 [60]. A relevant clinical finding is that tumors with Gleason score ≥ 8, i.e., less differentiated, tended to show a shorter interval to the emergence of small cell carcinoma [74], as the non-glandular tumor cells are closer to stem-like in lineage. On the other hand, stem-like cancer cells could be induced to differentiate into luminal-like cancer cells by prostate stromal mesenchyme cell factors [75]. From early tissue recombinant studies, stromal cells were found to determine the specificity of urologic organ development [76, 77]. Thus, prostate stromal cells would induce stem/progenitor cells, regardless of tissue origin, to differentiate into prostate; bladder stromal cells into bladder. This induction involves secreted factors and heterotypic cell contact. We identified proenkephalin (PENK, 267 aa) and stanniocalcin 1 (STC1, 247 aa) as prostate stromal-specific genes encoding

#### **Figure 9.**

*Lineage of prostate cancer cell types. Luminal-like, non-stem-like adenocarcinoma AGR2hi, scTF− B2Mhi and stem-like AGR2−/lo, scTF+ B2Mlo small cell carcinoma are related by de-differentiation with activation of scTF. The reverse of the process can be triggered by stromal cell factors such as PENK.*

**109**

scTF+

*Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2*

secreted hormones [78]. When cultured stem-like cells were incubated with prostate stromal cell conditioned media (containing stromal secreted molecules) PENK and STC1 were specifically induced in the differentiating stem cells. The resultant cells showed a change in colony morphology as well as one in transcriptome [79].

associated stromal cell conditioned media [79, 80]. The absence of PENK in the stroma of tumor foci suggests that it could be an underlying contributing cause of

PENK cDNA (803 bp) was cloned from microdissected benign prostate tissue [10], and was transfected into small cell carcinoma LuCaP 145.1 cells. LuCaP 145.1 is stem-like because of its expression of scTF, which are, as a quartet, absent in non-stem-like LuCaP adenocarcinoma lines [60, 81]. In addition, LuCaP 145.1 was found to share expression of other genes with stem cells, including the downregulation of β2-microglobulin (B2M) [60, 81, 82]. Forced expression of PENK in LuCaP 145.1 down-regulated the scTF, and up-regulated in tandem B2M [75].

to a phenotype (scTFlo/−B2Mhi) more characteristic of differentiated cancer cells was produced by PENK. In other words, PENK can counteract the activity of scTF. Therefore, PENK and other stromal factors could be envisioned as effective agents in differentiation therapy to maintain adenocarcinoma in the differentiated state

adenocarcinoma LuCaP 70CR (CR = castration resistant, a variant obtained from passages in castrated mice) was transfected by PENK. An increase in the expression of AGR2 was observed [75]. Increased production by LuCaP 70CR/PENK was validated by measurement of secreted AGR2 in the cell-free culture media [75]. The change in AGR2 expression indicated cancer cell differentiation induced from AGR2lo to AGR2hi by PENK in LuCaP 70CR. In contrast, AGR2 expression was down-regulated in reprogrammed LuCaP 70CR by scTF transfection to small cell carcinoma-like [60]. These results show that preventing or even reversing pros-

The cancer specificity of eAGR2 could allow us to develop a cancer vaccine in the future. Treated patients can be immunized by AGR2. Any emergent cells with eAGR2 expression are, by reasoning, cancerous and will be eliminated by a primed immune system. The result shown in **Figure 6** indicates no iAgr2-positive mouse organs were targeted outside non-specific background, which was non-overlapping in the study mice. Besides the bladder and lung, the intestinal tract containing Agr2+ mucus-producing cells [83] also did not show labeling. Secreted AGR2 is known to function in early development where it signals cell differentiation such as that described in limb regeneration of lower vertebrates [84]. Introduction of AGR2 was reported to accelerate wound healing through recruitment of fibroblasts and migration of keratinocytes [85]. So a possibility exists that AGR2-immunized patients would experience difficulty in tissue repair after damage. For these patients, one

To show the potential of an AGR2 cancer vaccine, we could immunize C57BL/6 mice with recombinant (r)AGR2 as was done in the generation of P1G4 and P3A5 (although a different mouse strain was used) [20]. After rAGR2 injection, mice are boosted at intervals and bled for ELISA testing of serum anti-AGR2 activity.

through stromal factor influence could keep anti-AGR2 therapy

The effect of PENK on AGR2 expression could be seen when scTF<sup>−</sup>

bladder stromal cell or PENK−

prostate cancer-

B2Mlo) exhibited by LuCaP 145.1

B2MhiAGR2hi to stem-like

B2Mhi

*DOI: http://dx.doi.org/10.5772/intechopen.96492*

Thus, a phenotypic change of stem-likeness (scTF+

tate cancer de-differentiation from luminal-like scTF−

**12.3 Cancer vaccine based on cancer-specificity of eAGR2**

could treat injuries with local administration of AGR2 protein.

a viable treatment option in the disease course.

PENK was not induced by PENK−

cancer development.

with high AGR2 expression.

B2MloAGR2−

#### *Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2 DOI: http://dx.doi.org/10.5772/intechopen.96492*

*Advances in Precision Medicine Oncology*

ing the AGR2+

cancer to anti-AGR2. For bladder cancer, we will carry out a similar study employ-

commonly used to treat this cancer, sometimes in combination with Gemcitabine [68]. In vitro-adapted CoCaB lines are similarly used for CDC and ADCC testing. Cancer treatment by antibody is well established and proven to be effective. One good example is trastuzumab to target HER2/EGFR (CD340) for a subset of breast cancer and non-small cell lung cancer [69, 70]. The major obstacle to more success in antibody therapy is the time-consuming need to discover TAA for each type of cancer that at a minimum is expressed by only a few normal cell types. eAGR2 is not only expressed in prostate cancer cells but also multiple tumor types so that anti-AGR2 would have a much wider application than just treating one or two cancers.

The use of anti-AGR2 might lead to the selection of AGR2lo/− non-adenocarcinoma cancer including small cell carcinoma. The introduction of newer antiandrogen therapies of late has led to an almost 20% patients presenting small cell carcinoma at treatment failure [71]. Of critical importance is finding a means to

**Figure 9** shows our model of prostate cancer differentiation relating luminal-

Stem-like cancer cells could arise from de-differentiation of luminal-like cancer cells. This process is akin to reprogramming of somatic cells via forced expression of a set of stem cell transcription factors (scTF) to induced pluripotent stem (iPS) cells [73]. We demonstrated that prostate adenocarcinoma cells could be so reprogrammed to stem-like, small cell carcinoma-like derivatives with scTF LIN28A, NANOG, POU5F1, SOX2 [60]. A relevant clinical finding is that tumors with Gleason score ≥ 8, i.e., less differentiated, tended to show a shorter interval to the emergence of small cell carcinoma [74], as the non-glandular tumor cells are closer to stem-like in lineage. On the other hand, stem-like cancer cells could be induced to differentiate into luminal-like cancer cells by prostate stromal mesenchyme cell factors [75]. From early tissue recombinant studies, stromal cells were found to determine the specificity of urologic organ development [76, 77]. Thus, prostate stromal cells would induce stem/progenitor cells, regardless of tissue origin, to differentiate into prostate; bladder stromal cells into bladder. This induction involves secreted factors and heterotypic cell contact. We identified proenkephalin (PENK, 267 aa) and stanniocalcin 1 (STC1, 247 aa) as prostate stromal-specific genes encoding

tumors.

*Lineage of prostate cancer cell types. Luminal-like, non-stem-like adenocarcinoma AGR2hi, scTF−*

*The reverse of the process can be triggered by stromal cell factors such as PENK.*

*B2Mlo small cell carcinoma are related by de-differentiation with activation of scTF.* 

Our experiments will demonstrate the validity of this claim.

**12.2 Lineage relationship between AGR2+**

adenocarcinoma to AGR2−

prevent the emergence of AGR2<sup>−</sup>

like AGR2+

CoCaB lines to test the ADC and P1G4 + cisplatin. Cisplatin is more

 **and AGR2−**

 **prostate cancer**

*B2Mhi and* 

more stem-like small cell carcinoma [72].

**108**

**Figure 9.**

*stem-like AGR2−/lo, scTF+*

secreted hormones [78]. When cultured stem-like cells were incubated with prostate stromal cell conditioned media (containing stromal secreted molecules) PENK and STC1 were specifically induced in the differentiating stem cells. The resultant cells showed a change in colony morphology as well as one in transcriptome [79]. PENK was not induced by PENK− bladder stromal cell or PENK− prostate cancerassociated stromal cell conditioned media [79, 80]. The absence of PENK in the stroma of tumor foci suggests that it could be an underlying contributing cause of cancer development.

PENK cDNA (803 bp) was cloned from microdissected benign prostate tissue [10], and was transfected into small cell carcinoma LuCaP 145.1 cells. LuCaP 145.1 is stem-like because of its expression of scTF, which are, as a quartet, absent in non-stem-like LuCaP adenocarcinoma lines [60, 81]. In addition, LuCaP 145.1 was found to share expression of other genes with stem cells, including the downregulation of β2-microglobulin (B2M) [60, 81, 82]. Forced expression of PENK in LuCaP 145.1 down-regulated the scTF, and up-regulated in tandem B2M [75]. Thus, a phenotypic change of stem-likeness (scTF+ B2Mlo) exhibited by LuCaP 145.1 to a phenotype (scTFlo/−B2Mhi) more characteristic of differentiated cancer cells was produced by PENK. In other words, PENK can counteract the activity of scTF. Therefore, PENK and other stromal factors could be envisioned as effective agents in differentiation therapy to maintain adenocarcinoma in the differentiated state with high AGR2 expression.

The effect of PENK on AGR2 expression could be seen when scTF<sup>−</sup> B2Mhi adenocarcinoma LuCaP 70CR (CR = castration resistant, a variant obtained from passages in castrated mice) was transfected by PENK. An increase in the expression of AGR2 was observed [75]. Increased production by LuCaP 70CR/PENK was validated by measurement of secreted AGR2 in the cell-free culture media [75]. The change in AGR2 expression indicated cancer cell differentiation induced from AGR2lo to AGR2hi by PENK in LuCaP 70CR. In contrast, AGR2 expression was down-regulated in reprogrammed LuCaP 70CR by scTF transfection to small cell carcinoma-like [60]. These results show that preventing or even reversing prostate cancer de-differentiation from luminal-like scTF− B2MhiAGR2hi to stem-like scTF+ B2MloAGR2− through stromal factor influence could keep anti-AGR2 therapy a viable treatment option in the disease course.

#### **12.3 Cancer vaccine based on cancer-specificity of eAGR2**

The cancer specificity of eAGR2 could allow us to develop a cancer vaccine in the future. Treated patients can be immunized by AGR2. Any emergent cells with eAGR2 expression are, by reasoning, cancerous and will be eliminated by a primed immune system. The result shown in **Figure 6** indicates no iAgr2-positive mouse organs were targeted outside non-specific background, which was non-overlapping in the study mice. Besides the bladder and lung, the intestinal tract containing Agr2+ mucus-producing cells [83] also did not show labeling. Secreted AGR2 is known to function in early development where it signals cell differentiation such as that described in limb regeneration of lower vertebrates [84]. Introduction of AGR2 was reported to accelerate wound healing through recruitment of fibroblasts and migration of keratinocytes [85]. So a possibility exists that AGR2-immunized patients would experience difficulty in tissue repair after damage. For these patients, one could treat injuries with local administration of AGR2 protein.

To show the potential of an AGR2 cancer vaccine, we could immunize C57BL/6 mice with recombinant (r)AGR2 as was done in the generation of P1G4 and P3A5 (although a different mouse strain was used) [20]. After rAGR2 injection, mice are boosted at intervals and bled for ELISA testing of serum anti-AGR2 activity.

IgM-to-IgG switch is monitored. Once an adequate antibody titer is measured, syngeneic mouse bladder cancer cells MB49 [86] are implanted *vs*. into control animals without AGR2 immunization. MB49 was derived from DMBA-transformed (presumably iAgr2+ ) bladder epithelial cells of C57BL. Whether these cancer cells express eAgr2 will be determined. If eAgr2 is not detected, we can transfect these cells with our AGR2 plasmid construct (550-bp full length cDNA cloned from prostate cancer tissue). AGR2− LNCaP cells when transfected by this plasmid produced secreted and cell surface AGR2 [81]. Note we do not need to transfect the murine Agr2 gene because the antibodies produced would recognize both human AGR2 and mouse Agr2 as shown for P3A5. We expect that the immunized mice would show no tumor growth. AGR2 vaccination will, in principle, prevent recurrence and metastasis.
